Parathyroid hormone receptor activation and stem and progenitor cell expansion

ABSTRACT

The invention relates to methods for manipulating hematopoietic stem or progenitor cells, mesenchymal stem cells, epithelial stem cells, neural stem cells and related products through activation of the PTH/PTHrP receptor in neighboring cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/621,325,filed Nov. 18, 2009, issuing, which is a division of U.S. applicationSer. No. 11/268,971, filed Nov. 8, 2005, issued as U.S. Pat. No.7,635,477 on Dec. 22, 2009, which is a continuation-in-part of U.S.application Ser. No. 10/521,971, filed on Jan. 21, 2005, issued as U.S.Pat. No. 7,429,383 on Sep. 30, 2008, which is the U.S. national phasepursuant to 35 U.S.C. §371 of International Appln. No. PCT/US03/023425,filed Jul. 25, 2003, which claims priority to U.S. ProvisionalApplication Ser. No. 60/398,801, filed Jul. 25, 2002. U.S. applicationSer. No. 11/268,971, filed Nov. 8, 2005, also claims priority to U.S.Provisional Application Ser. No. 60/648,216, filed on Jan. 28, 2005 andU.S. Provisional Application Ser. No. 60/626,671, filed Nov. 11, 2004.The contents of each of the aforementioned patent applications areexpressly incorporated herein by reference.

Each of the applications and patents cited in this text, as well asdocuments or references cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited document”) and each of the PCT and foreign applications andpatents, and each of the documents cited or referenced in each of theapplication cited documents, are hereby expressly incorporated herein byreference, and may be employed in the practice of the invention. Moregenerally, documents or references are cited in this text, either in aReference List before the claims, or in the text itself; and, each ofthese documents or references (“herein cited references”), as well aseach document or reference cited in each of the herein cited references(including any manufacturer's specifications, instructions, etc.), ishereby expressly incorporated herein by reference. Documentsincorporated by reference into this text or any teaching therein can beused in the practice of this invention.

GOVERNMENT SUPPORT

The work leading to the present invention was funded in part bycontract/grant numbers HL65909, CA86355, DK60317, and AR44855, from theUnited States National Institutes of Health. Accordingly, the UnitedStates Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

Circulating blood cells, such as erythrocytes, leukocytes, platelets andlymphocytes, arise from the terminal differentiation of precursor cells,in a process referred to as hematopoiesis. In fetal life, hematopoiesisoccurs throughout the reticular endothelial system. In the normal adult,terminal differentiation of the precursor cells occurs exclusively inthe marrow cavities of the axial skeleton, with some extension into theproximal femora and humeri. These precursor cells, in turn, derive fromimmature cells, called progenitors, stem cells or hematopoietic cells.

Hematopoietic progenitor cells have therapeutic potential as a result oftheir capacity to restore blood and immune cell function in transplantrecipients as well as their potential ability to generate cells forother tissues such as brain, muscle and liver (Choi, 1998 Biochem CellBiol 76, 947-56; Eglitis and Mezey, 1997 Proc Natl Acad Sci USA 94,4080-5; Gussoni et al., 1999 Nature 401, 390-4; Theise et al., 2000Hepatology 32, 11-6). Human autologous and allogeneic bone marrowtransplantation methods are currently used as therapies for diseasessuch as leukemia, lymphoma, and other life-threatening diseases. Forthese procedures a large amount of donor bone marrow must be isolated toensure that there are enough cells for engraftment. Hematopoieticprogenitor cell expansion for bone marrow transplantation is a potentialmethod for generating human long-term bone marrow cultures for thesetherapeutic utilities. Several studies have reported the isolation andpurification of hematopoietic progenitor cells (see, e.g., U.S. Pat. No.5,061,620), but none of these methods have been overwhelminglysuccessful.

Determining the basis for progenitor cell localization is important tomaximizing the therapeutic potential of those cells. During development,hematopoiesis translocates from the fetal liver to the bone marrow,which then remains the site of hematopoiesis throughout adulthood. Oncehematopoiesis has been established in the bone marrow, the hematopoieticprogenitor cells are not distributed randomly throughout the bonecavity. Instead, the hematopoietic progenitor cells are found in closeproximity to the endosteal surfaces (Lord et al., 1975, Blood, 46:65-72;Gong et al., 1978, Science, 199:1443-1445), an observation recentlyconfirmed when injected purified hematopoietic progenitor cells werefound to preferentially localize to the endosteal surfaces approximately10 hours following injection (Nilsson, et al., 2001, Blood,97:2293-2299). The more mature progenitor cells (as measured by theirCFU-C activity) increased in number as the distance from the bonesurface increased. Finally, as the central longitudinal axis of the boneis approached, it has been shown that terminal differentiation of maturecells occurs (Lord et al., 1975, Blood, 46:65-72; Cui et al., 1996, CellProlif., 29:243-257; Lord et al., 1990, Int. J. Cell Clon., 8:317-331).

Given the relationship between the hematopoietic progenitor cells andthe endosteal surfaces of the bone, one cell type that has beenimplicated in playing a role in hematopoiesis is the osteoblast(Taichman and Emerson, 1998, Stem Cells, 16:7-15). Osteoblastic cellsare skeletal cells responsible for the production and mineralization ofbone matrix, in response to local and hormonal stimuli (Ducy, et al.,2000, Science, 289:1501-1504). In addition, these cells regulate boneremodeling by modulating the formation and activity of osteoclasts,bone-resorbing cells of hematopoietic origin, through theRANK/RANK-Ligand system (Teitelbaum et al., 2000, Science,289:1504-1508). Studies have demonstrated that osteoblastic cells cansupport the growth of primitive hematopoietic cells, through the releaseof G-CSF and other growth factors (Taichman and Emerson, 1994, J. Exp.Med., 179:1677-1682; Taichman et al., 1996, Blood, 87:518-524; Taichmanet al., 2001, Br. J. Haematol., 112:438-448).

The ability to manipulate progenitor cells could improve the efficiencyof engraftment of transplanted cells. Currently, transplantationtechniques are extremely inefficient. In view of their enormoustherapeutic potential relatively little is known about how hematopoieticprogenitor cells are regulated, e.g., what factors cause celllocalization, expansion, etc. Some studies have suggested thatprogenitor cell localization into the bone marrow space is chemokinedependent. For instance, the absence of either SDF-1 or its receptor,CXCR-4, was found to preclude localization of hematopoiesis in the bonemarrow in developing mice (Nagasawa et al., 1996, Nature, 382:635-8; Suet al., 1999, J. Immunol., 162:7128-7132; Zou et al., 1998, Nature,393:595-9). In addition, manipulation of CXCR-4 alters the homing andretention of progenitors in adult mice further supporting its criticalrole (Ma et al., 1999, Immunity, 10:463-71; Peled et al., 1999, Science,283:845-8). Selectins and integrins are also believed to participate inthis process and have been identified as mediators of retention oradhesion of primitive cells to bone marrow in vivo or in vitro(Greenberg et al., 2000, Blood, 95:478-86; Naiyer et al., 1999, Blood,94:4011-9; Rood et al., 1999, Exp. Hematol., 27:1306-14; van der Loo etal., 1998, J. Clin. Invest. 102:1051-61; Williams et al., 1991, Nature,352:438-41; Zanjani et al., 1999, Blood, 94:2515-22). These studies,however, have not provided a complete understanding of progenitor celllocalization.

Understanding exogenous signaling molecules which may contribute to theexpansion of the progenitor cell population is important to definingtherapeutic procedures.

SUMMARY OF THE INVENTION

The invention relates in some aspects to methods for manipulating stemand progenitor cells. It has been discovered, surprisingly, thatactivation of the Parathyroid Hormone/Parathyroid Hormone-relatedProtein (PTH/PTHrP) receptor in cells forming a microenvironmentaccording to the invention leads to an enhancement in the growth(including increase in self-renewal/number increase) and/or maintenanceof progenitor and stem cells (e.g., hematopoietic stem cells,hematopoietic progenitor cells, mesenchymal stem cells, epithelial stemcells, neural stem cells).

In one aspect the invention relates to a method for enhancing the growthor maintenance of hematopoietic stem or progenitor cells. The methodinvolves, contacting a cell expressing a PTH/PTHrP receptor with anagent that activates the PTH/PTHrP receptor in an amount effective tosupport the growth or maintenance of hematopoietic stem or progenitorcells. In important embodiments, the cell expressing a PTH/PTHrPreceptor is present in the immediate vicinity of a hematopoietic stem orprogenitor cell. In one embodiment, the cell expressing a PTH/PTHrPreceptor is a lymphoreticular stromal cell. In a further embodiment, thecell expressing a PTH/PTHrP receptor is a hematopoietic progenitor cell.Contacting of the cell expressing a PTH/PTHrP receptor with an agentthat activates the PTH/PTHrP receptor may occur in vitro or in vivo. Inimportant embodiments, the agent that activates the PTH/PTHrP receptoris PTH (including recombinant synthetic human PTH (1-34) and active PTHfragments), a PTH analogue, or a PTH/PTHrP receptor agonist. The growthor maintenance of hematopoietic progenitor cells may occur in vitro orin vivo.

In another aspect of the invention a method for inducing hematopoieticstem or progenitor cell self-renewal, is provided. The method involvesco-culturing a hematopoietic stem or progenitor cell with a cellexpressing a PTH/PTHrP receptor, and contacting the cell expressing aPTH/PTHrP receptor with an agent that activates the PTH/PTHrP receptorto induce self-renewal of the hematopoietic stem or progenitor cell. Theco-culturing may occur in vitro or ex vivo.

In a further aspect of the invention a method for enhancing the growthor maintenance of hematopoietic stem or progenitor cells in a subject,is provided. The method involves administering to a subject in need ofsuch treatment an agent that activates the PTH/PTHrP receptor in cellsof the subject expressing the PTH/PTHrP receptor, in an amount effectiveto support the growth or maintenance of hematopoietic stem or progenitorcells. In some embodiments, the cell expressing a PTH/PTHrP receptor isa lymphoreticular stromal cell. In certain embodiments, the cellexpressing a PTH/PTHrP receptor is a hematopoietic stem or progenitorcell. In important embodiments, the agent that activates the PTH/PTHrPreceptor is PTH, a PTH analogue, or a PTH/PTHrP receptor agonist. Infurther important embodiments, the subject in need of such treatment isa bone marrow donor. The bone marrow donor may have donated bone marrow,or has yet to donate bone marrow. In certain embodiments, the subject inneed of such treatment is a bone marrow transplant recipient. In oneembodiment, the subject in need of such treatment is a subject havinghematopoietic progenitor cells under environmental stress. Environmentalstresses include increased temperatures (e.g., fever), physical trauma,oxidative, osmotic and chemical stress (e.g. a chemotherapeutic agent),and/or irradiation (e.g. ultra-violet (UV), X-ray, gamma, alpha, or betairradiation).

In further embodiments the subject in need of such treatment is asubject having immune system deficiencies. Immune system deficienciesinclude subjects with chronic infections, subjects treated withradiation or chemotherapy, subjects with abnormally low CD4 cell counts,subjects with genetic immune deficiencies. The subject can also be asubject with any one or more categories of hematopoietic cell deficiencysuch as abnormally low monocytes, macrophages, neutrophils, T-cells,B-cells, erythrocytes, platelets, basophils.

In a further aspect of the invention a method for providinghematopoietic cells to a subject in need thereof, is provided. Themethod involves administering an agent that activates a PTH/PTHrPreceptor in cells of the subject expressing the PTH/PTHrP receptor in anamount effective to increase hematopoietic stem or progenitor cellproduction. In some embodiments, the cell expressing a PTH/PTHrPreceptor is a lymphoreticular stromal cell. In certain embodiments, thecell expressing a PTH/PTHrP receptor is a hematopoietic stem orprogenitor cell. In important embodiments, the agent that activates thePTH/PTHrP receptor is PTH, a PTH analogue, or a PTH/PTHrP receptoragonist. In further important embodiments, the subject in need of suchtreatment has received, will receive or is concurrently receivingchemotherapy or radiation therapy for cancer. The subject can have adisorder including but not limited to myeloma, non-Hodgkin's lymphoma,Hodgkins lyphoma and leukaemia. The subject can have a disordercharacterized by a lack of functional blood cells, including but notlimited to a platelet deficiency, anemia (e.g., aplastic anemia, sicklecell anemia, fanconi's anemia and acute lymphocytic anemia) andneutropenia. The subject can have a disorder characterized by a lack offunctional immune cells, including but not limited to, lymphocytopenia,lymphorrhea, lymphostasis and AIDS. The subject can also be a stem celldonor. In important embodiments, the subject in need of such treatmenthas received, will receive or is concurrently receiving animmuno-suppressive drug. In further important embodiments, the subjectin need of such treatment has received, will receive or is concurrentlyreceiving G-CSF.

According to another aspect of the invention a method for enhancingmobilization of hematopoietic stem or progenitor cells, is provided. Themethod involves administering to a subject in need of such treatment anagent that activates a PTH/PTHrP receptor in an amount sufficient toenhance mobilization of hematopoietic stem or progenitor cells in thesubject. In important embodiments, the subject is a bone marrow donor.

In a further aspect of the invention a method for increasing the ratioof normal to abnormal hematopoietic cells in a subject in need thereof,is provided. The method involves contacting a population of cellsexpressing a PTH/PTHrP receptor in the subject with an agent thatactivates the PTH/PTHrP receptor in an amount effective to expand apopulation of hematopoietic stem or progenitor cells, thereby increasingthe ratio of normal to abnormal hematopoietic cells in the subject. Insome embodiments, the population of cells expressing a PTH/PTHrPreceptor are present in the immediate vicinity of the population ofhematopoietic stem or progenitor cells. The population of cellsexpressing a PTH/PTHrP receptor can be but are not limited toosteoblasts, lymphoreticular stromal cells, and a mixture of osteoblastsand lymphoreticular stromal cells. In important embodiments, theabnormal cells are leukemic cells (e.g., lymphoblastic) or pre-leukemiccells (e.g., myelodysplasic cells). In important embodiments, the agentthat activates the PTH/PTHrP receptor is PTH, a PTH analogue, or aPTH/PTHrP receptor agonist. In further important embodiments, thesubject in need of such treatment has or is at risk of having leukemia,wherein the leukemia is chronic (e.g., chronic myeloid, chronicmyelogenous or chronic granulocytic leukemia) or the leukemia is acute(e.g., acute lymphoblastic leukemia or acute nonlymphoblastic leukemia).

In a further aspect of the invention a method for treating a subjecthaving or at risk of having leukemia, is provided. The method involvesadministering to the subject a PTH, a PTH analogue, or a PTH/PTHrPreceptor agonist in an amount effective to increase the amount of normalhematopoietic stem and progenitor cells; and decreasing the amount ofleukemic or pre-leukemic cells, thereby treating a subject having or atrisk of having leukemia. In important embodiments, the subject in needof such treatment has or is at risk of having leukemia, wherein theleukemia is chronic (e.g., chronic myeloid, chronic myelogenous orchronic granulocytic leukemia) or the leukemia is acute (e.g., acutelymphoblastic leukemia or acute nonlymphoblastic leukemia).

In a further aspect of the invention a method for decreasing the amountof abnormal hematopoietic cells in a subject in need thereof, isprovided. The method involves administering to the subject a PTH, a PTHanalogue, or a PTH/PTHrP receptor agonist in an amount effective toincrease the amount of normal hematopoietic stem and progenitor cells inthe subject, thereby decreasing the amount of abnormal hematopoieticcells in the subject. In important embodiments, the abnormal cells areleukemic cells (e.g., lymphoblastic) or pre-leukemic cells (e.g.,myelodysplasic cells). In important embodiments, the agent thatactivates the PTH/PTHrP receptor is PTH, a PTH analogue, or a PTH/PTHrPreceptor agonist. In further important embodiments, the subject in needof such treatment has or is at risk of having leukemia, wherein theleukemia is chronic (e.g., chronic myeloid, chronic myelogenous orchronic granulocytic leukemia) or the leukemia is acute (e.g., acutelymphoblastic leukemia or acute nonlymphoblastic leukemia).

According to a further aspect, the invention provides an isolatedpopulation of cells treated with PTH. The population of cells ispreferably a stromal cell population. The cells can be ex vivo cellsisolated from a subject. Alternatively, the cells can be in vitrocultured cells. In one embodiment, the isolated cells are homogeneous.In an alternative embodiment, the isolated cells are heterogeneous andinclude two or more cell types. One of the cell types is preferably astromal cell.

Methods of the invention can be applied to non-hematopoietic stem andprogenitor cells.

In another aspect, a method for enhancing the growth or maintenance ofmesenchymal stem cells, is provided. The method involves, contacting acell expressing a PTH/PTHrP receptor with an agent that activates thePTH/PTHrP receptor in an amount effective to support the growth ormaintenance of mesenchymal stem cells. In important embodiments, thecell expressing a PTH/PTHrP receptor is present in the immediatevicinity of a mesenchymal stem cell. In one embodiment, the cellexpressing a PTH/PTHrP receptor is a bone (e.g., osteoblast) breast(e.g., mammary cells), skin (e.g., keratinocytes and fibroblasts),epithelial, lung (e.g., alveolar cells), urogenital, or gastrointestinalcell. The growth or maintenance of mesenchymal stem cells may occur invitro or in vivo.

In yet another aspect, a method for increasing the ratio of normal toabnormal bone, mammary, skin, lung, urogenital or gastrointestinal cellsin a subject in need thereof, is provided. The method involvescontacting a population of cells expressing a PTH/PTHrP receptor in thesubject with an agent that activates the PTH/PTHrP receptor in an amounteffective to expand a population of mesenchymal stem or progenitorcells, thereby increasing the ratio of normal to abnormal bone, mammary,skin, lung, urogenital or gastrointestinal cells in the subject.

In yet another aspect, a method for enhancing the growth or maintenanceof epithelial stem cells, is provided. The method involves, contacting acell expressing a PTH/PTHrP receptor with an agent that activates thePTH/PTHrP receptor in an amount effective to support the growth ormaintenance of epithelial stem cells. In important embodiments, the cellexpressing a PTH/PTHrP receptor is present in the immediate vicinity ofa epithelial stem cell. In one embodiment, the cell expressing aPTH/PTHrP receptor is a breast (e.g., mammary cells), skin (e.g.,keratinocytes, fibroblasts, hair follicle cells), epithelial, lung(e.g., alveolar cells) urogenital or gastrointestinal. The growth ormaintenance of epithelial stem cells may occur in vitro or in vivo.

In yet another aspect, a method for increasing the ratio of normal toabnormal mammary, skin, lung, urogenital or gastrointestinal cells in asubject in need thereof, is provided. The method involves contacting apopulation of cells expressing a PTH/PTHrP receptor in the subject withan agent that activates the PTH/PTHrP receptor in an amount effective toexpand a population of epithelial stem or progenitor cells, therebyincreasing the ratio of normal to abnormal mammary, skin, lung,urogenital or gastrointestinal cells in the subject.

In yet another aspect, a method for enhancing the growth or maintenanceof neural stem cells, is provided. The method involves, contacting acell expressing a PTH/PTHrP receptor with an agent that activates thePTH/PTHrP receptor in an amount effective to support the growth ormaintenance of neural stem cells. In important embodiments, the cellexpressing a PTH/PTHrP receptor is present in the immediate vicinity ofa neural stem cell. In one embodiment, the cell expressing a PTH/PTHrPreceptor is a astrocyte, oligodendrocyte, glial cell, GABAergic neuronor dopaminergic neuron. In another embodiment, the cell expressing aPTH/PTHrP receptor is located in a particular anatomical region of thebrain, such as a cell of the cerebellum, (e.g., a purkinje cell, agranule cell), telencephalon, diencephalons, mesencephalon, medulla,pons, thalamus, hippocampus, trigeminal ganglion or leptomeninges. Thegrowth or maintenance of neural stem cells may occur in vitro or invivo.

In yet another aspect, a method for increasing the ratio of normal toabnormal neural cells in a subject in need thereof, is provided. Themethod involves contacting a population of cells expressing a PTH/PTHrPreceptor in the subject with an agent that activates the PTH/PTHrPreceptor in an amount effective to expand a population of neural stem orprogenitor cells, thereby increasing the ratio of normal to abnormalneural cells in the subject.

In yet another aspect, the present invention provides a kit forenhancing the growth or maintenance of hematopoietic stem or progenitorcells, epithelial stem cells or mesenchymal stem cells and instructionsfor using an agent that activates the PTH/PTHrP receptor to enhance thegrowth or maintenance of the cells in accordance with the methodsdescribed herein.

In yet another aspect, a method of identifying a cellular product thatincreases a population of stem or progenitor cells is provided, themethod comprising the steps of:

-   -   a) contacting a cell expressing a PTH/PTHrP receptor with an        agent that activates the PTH/PTHrP receptor;    -   b) collecting proteins or mRNA encoding proteins produced by the        cell expressing a PTH/PTHrP receptor in response to the agent of        step a);    -   c) contacting a stem or progenitor cell with one or more        proteins of step b);    -   d) measuring a physiologic effect exhibited by the stem or        progenitor cell; and    -   e) isolating one or more proteins associated with the        physiologic effect,

wherein the physiologic effect comprises increased replication of thestem or progenitor cells.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Figure showing hPTH/PTHrP receptor construct.

FIG. 2. a) Graph showing the frequency of Sca-1⁺lin⁻ subpopulation cellsfrom total bone marrow mononuclear cells. b) Graph showing hematopoieticstem cell frequency of lin⁻ fraction of bone marrow mononuclear cells.c) Plot showing proportion of Sca-1 lin⁻ cells in G₀ vs G₁ phase. d)Graph showing frequency of hematopoietic stem cells using the CFU-Cassay.

FIG. 3. a) Graph showing support of stromal cells from transgenic mice.b) Plot showing the level of NICD in lin⁻Sca-1⁺c-Kit⁺ hematopoietic stemcells.

FIG. 4. Graph showing LTC-IC assay under non-contact culture conditions.

FIG. 5. a) Graph showing LTC-IC assay of C57B1/6 stroma expansion in theabsence or presence of PTH. b) Photo showing alkaline phosphatasepositive cells. c) Graph showing inhibition of LTC-IC in the presence orabsence of PTH. d) Graph showing percentage of lin⁻Sca-1⁺c-Kit⁺ cells inbone marrow in mock injected and PTH injected mice. e) Graph showingincrease of LTC-ICs in bone marrow mononuclear cells in mock injectedand PTH injected mice. f) Graph showing percentage of CD45.2⁺ cells inbone marrow in mock injected and PTH injected mice. g) Graph showingCFU-Cs in bone marrow mononuclear cells in mock injected and PTHinjected mice.

FIG. 6. Plot showing percent survival of mock injected and PTH injectedmice.

FIGS. 7A-G. Depict the treatment protocol (FIG. 7A) and: analysis of HSCin peripheral blood following chemotherapy, PTH treatment and G-CSFtreatment in terms of white blood count (WBC, FIG. 7B), neutrophilscount (NE, FIG. 7C), hemoglobin concentration (Hb, FIG. 7D) and plateletcount (Plt, FIG. 7E); and analysis of HSC in bone marrow (FIG. 7F) andperipheral blood (FIG. 7G) with G-CSF upon prior and concurrenttreatment with PTH.

FIG. 8. Depicts the structure of the amino acid peptide derivative ofPTH Leu²⁷cyclo[Glu²²-Lys²⁶]-hPTH(1-31)-NH₂ (Ostabolin-C™) (SEQ ID NO:1).

FIG. 9. a) Graph showing tracking of GFP+ cells in total cellpopulation. b) Bar graphs showing percent leukemia (GFP+) cells overtime in the marrow (above) and blasts per 10⁴ over time in theperiphery.

FIG. 10. a) Schematic depiction of leukemia outgrowth in“niche-stimulated” animal study. b) Bar graph showing percent GFP+ cellsscored after bone marrow transplant and harvest of afore-mentionedstudy. c) Histological images of 10-micron bone sections fromtransplanted mock-treated vs. PTH-treated animals.

FIG. 11. Bar graph showing unchanged leukemia inhibition effect asobserved for mock-treated vs. PTH-treated animals with a six-foldincrease in HSCs and with no such increase.

FIG. 12. a) Schematic depiction of osteoblast/C1498/GFP leukemia cellco-culture experiment. b) FACS plot showing osteoblasts isolated on thebasis of CD45 and PTH receptor. c) Alkaline phosphatase staining ofCD45⁻, PTH receptor⁺ population of osteoblasts. d) Further schematicdepiction of osteoblast/C1498/GFP leukemia cell co-culture experiment.e) Bar graph showing total cell number vs. fibroblasts alone andfibroblasts with osteoblasts.

FIG. 13. a) Bar graph showing percent annexin-positive cells uponculture with varying doses of recombinant osteopontin. b) Bar graphshowing cell number×10⁴ for leukemia cells under different cultureconditions.

DETAILED DESCRIPTION OF THE INVENTION

New methods for manipulating progenitor and stem cells have beenidentified according to the invention. These methods and relatedproducts have great therapeutic and research value. For instance,hematopoietic progenitor cells are used for transplantation tosupplement the immune system of immune deficient patients. These cellshave many additional therapeutic uses. Prior to the invention, however,the ability to isolate and purify hematopoietic progenitor cells hasbeen limited. These cells reside in the bone marrow, making theirisolation a technically complex procedure. Additionally, there are notmany commercially viable methods for identifying these cells in asample. The invention has solved many of these problems.

According to the invention, a method for enhancing the growth ormaintenance of stem or progenitor cells is provided. The methodinvolves, contacting a cell expressing a PTH/PTHrP receptor with anagent that activates the PTH/PTHrP receptor in an amount effective tosupport the growth or maintenance of stem or progenitor cells (e.g.,hematopoietic stem cells, hematopoietic progenitor cells, mesenchymalstem cells, epithelial stem cells, neural stem cells).

As used herein, “enhancing the growth or maintenance” refers topromoting, increasing or enhancing the condition of the stem orprogenitor cells, including the survival and differentiation capacity ofthe cells.

“Stem cells” as used herein refer to immature cells having the capacityto self-renew and to differentiate into the more mature cells (alsodescribed herein as “progeny”). Progenitor cells also have the capacityto self-renew and to differentiate into more mature cells, but arecommitted to a lineage (e.g., hematopoietic progenitors are committed tothe blood lineage), whereas stem cells are not necessarily so limited.For the purposes of this disclosure, progenitor cells can beinterchangeably described as “stem cells” throughout the specification.

Methods of the invention further provide a means for expansion ofnon-hematopoietic stem and progenitor cells, such as epithelial,mesenchymal, and neural stem cells. Growth and expansion of such stemcell populations can improve tissue quality among multiple organsystems, including, for example, neural, breast, skin, respiratory,muscle, bone, urogenital or gastrointestinal systems. Furthermore,increasing the amount of epithelial, mesenchymal, or neural stem andprogenitor cells in a subject having abnormal cells (e.g., malignantcells) of the same origin can increase the ratio of normal to abnormalcells.

Accordingly, stem cell populations that can benefit from methods of theinvention include mesenchymal stem cells. Mesenchymal stem cells arebelieved to migrate out of the bone marrow, to associate with specifictissues, where they will eventually differentiate into multiplelineages. Enhancing the growth and maintenance of mesenchymal stemcells, in vitro or ex vivo will provide expanded populations that can beused to generate new tissue, including breast, skin, muscle,endothelium, bone, respiratory, urogenital, gastrointestinal connectiveor fibroblastic tissues.

Mesenchymal stem cells, or “MSCs” are well known in the art. MSCs,originally derived from the embryonal mesoderm and isolated from adultbone marrow, can differentiate to form muscle, bone, cartilage, fat,marrow stroma, and tendon. During embryogenesis, the mesoderm developsinto limb-bud mesoderm, tissue that generates bone, cartilage, fat,skeletal muscle and endothelium. Mesoderm also differentiates tovisceral mesoderm, which can give rise to cardiac muscle, smooth muscle,or blood islands consisting of endothelium and hematopoietic progenitorcells. Primitive mesodermal or MSCs, therefore, could provide a sourcefor a number of cell and tissue types. A number of MSCs have beenisolated. (See, for example, Caplan, A., et al., U.S. Pat. No.5,486,359; Young, H., et al., U.S. Pat. No. 5,827,735; Caplan, A., etal., U.S. Pat. No. 5,811,094; Bruder, S., et al., U.S. Pat. No.5,736,396; Caplan, A., et al., U.S. Pat. No. 5,837,539; Masinovsky, B.,U.S. Pat. No. 5,837,670; Pittenger, M., U.S. Pat. No. 5,827,740;Jaiswal, N., et al., (1997). J. Cell Biochem. 64(2):295-312; CassiedeP., et al., (1996) J Bone Miner Res. 9:1264-73; Johnstone, B., et al.,(1998) Exp Cell Res. 1:265-72; Yoo, et al., (1998) J Bon Joint Surg Am.12:1745-57; Gronthos, S., et al., (1994) Blood 84:4164-73; Pittenger, etal., (1999) Science 284:143-147). This cell is capable ofdifferentiating into a number of cell types of mesenchymal origin. MSCscan also differentiate into endodermal and ectodermal, including neural,lineages.

Stem cell populations that can benefit from methods of the inventionalso include epithelial stem cells. The epithelium is the membranouscellular tissue that covers the surface or lines a tube or cavity of ananimal body. The epithelium serves to enclose and protect the otherparts of the body and may produce secretions and excretions and may beassociated with assimilation as seen in the gastrointestinal tract. Theepithelium is one of the four primary tissues of the body, whichconstitutes the epidermis and the lining of respiratory, digestive andgenitourinary passages.

Epithelial stem cells are also well-known in the art. Epithelial stemcells are cells that are long-lived, relatively undifferentiated, have agreat potential for cell division, and are ultimately responsible forthe homeostasis of epithelium. Cells of this type include, but are notlimited to, those described in U.S. Pat. Nos. 5,556,783; 5,423,778;Rochat et al., Cell 76:1063 (1994); Jones et al. Cell 73:713 (1993);Jones et al., Cell 80:83 (1995)) and Slack, Science 287:1431-1433(2000).

Skin is one source of epithelial stem cells. Human skin consists of anouter layer of epithelial cells, the epidermis, and an inner layer ofsupporting tissue, the dermis. The dermis is a well vascularized tissuethat provides support for the epidermis. The dermis containsfibroblasts, which produce various elements of the connective tissue,including the extracellular matrix proteins such as collagens,fibronectin and elastin, which contribute to the strength andflexibility of the skin. The skin also contains various accessory organssuch as hair follicles and sweat glands. The epidermis is composed of acontinually renewing stratified layer of epithelial cells, calledkeratinocytes. The basal layer of the epidermis contains epithelial stemcells that divide and give rise to the keratinocytes (among other celltypes), which produce keratin as they differentiate and are “pushed” tothe surface of the epidermis. Epithelial stem cells (“ESCs”) can beobtained from tissues such as the skin and the lining of the gut byknown procedures, and can be grown in tissue culture (Rheinwald, 1980,Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo Clinic Proc.61:771).

Stem cell populations that can benefit from methods of the inventionalso include neural stem cells. Enhancing the growth and maintenance ofneural stem cells, in vitro or ex vivo will provide expanded populationsthat can be used to generate neural tissue, including astrocytes,oligodendrocytes glial cells, GABAergic and dopaminergic neurons.

Neural stem cells are known in the art (Gage F. H. (2000) Science287:1433:1438; Svendsen C. N. et al, (1999) Brain Path 9:499-513; OkabeS. et al. (1996) Mech Dev 59:89-102.) It was previously believed thatthe adult brain no longer contained cells with stem cell potential.However, several studies in rodents, and more recently, non-humanprimates and humans, have shown that stem cells persist in adult brain.These stem cells can proliferate in vivo and continuously regenerate atleast some neuronal cells in vivo. When cultured ex vivo, neural stemcells can be induced to proliferate, as well as to differentiate intodifferent types of neurons and glial cells. When transplanted into thebrain, neural stem cells can engraft and generate neural cells and glialcells.

Neural stem cells have been identified in the sub-ventricular zone andthe hippocampus of the adult mammalian brain (Ciccolini et al., (1998) JNeuroscience 18: 7869-7880; Palmer et al., (1999) J. Neurosci.19:8487-97; Reynolds and Weiss, (1992) Science 255:1707-10; Vescovi etal., (1999) Exp Neurol 156:71-83) and can also be present in theependyma and other presumed non-neurogenic areas of the brain (Doetschet al., (1999) Cell 97:703-716; Johansson et al., (1999) Cell 96, 25-34;Palmer et al., (1999) J. Neurosci. 19:8487-97). Fetal or adultbrain-derived neural stem cells can be expanded ex vivo and induced todifferentiate into astrocytes, oligodendrocytes and functional neurons(Ciccolini et al., (1998) J Neuroscience 18: 7869-7880; Johansson etal., (1999) Cell 96, 25-34; Palmer et al., (1999) J Neurosci.19:8487-97; Reynolds et al., (1996) Dev Biot 175:1-13; Ryder et al.,(1990) J Neurobiol 21: 356-375; Studer et al., (1996) Exp Brain Res 108,328-36; Vescovi et al., (1993) Neuron 11, 951-66). In vivo,undifferentiated neural stem cells cultured for variable amounts of timeeventually differentiate into glial cells, GABAergic and dopaminergicneurons (Flax et al., (1998) Nature Biotechnol 16:1033-1038; Gage etal., (1995) Proc Natl Adad Sci USA 92:11879-83; Suhonen et al., (1996)Nature 383:624-7).

Cells expressing a PTH/PTHrP receptor can be present in the immediatevicinity of neural stem cells. For example, the cell expressing aPTH/PTHrP receptor can be located in a particular anatomical region ofthe brain, such as a cell of the cerebellum, (e.g., a purkinje cell, agranule cell), telencephalon, diencephalons, mesencephalon, medulla,pons, thalamus, hippocampus, trigeminal ganglion or leptomeninges(Weaver et al. (1995) Mol. Brain. Res. 28:296.

In another aspect, a method for enhancing the growth or maintenance ofhematopoietic stem or progenitor cells is provided. The method involves,contacting a cell expressing a PTH/PTHrP receptor with an agent thatactivates the PTH/PTHrP receptor in an amount effective to support thegrowth or maintenance of hematopoietic stem or progenitor cells.

It has been discovered according to some aspects of the invention thatactivation of the parathyroid hormone/parathyroid hormone-relatedprotein receptor (PTH/PTHrP) results in enhancing the growth (includingincrease in self-renewal/number increase) or maintenance ofhematopoietic stem or progenitor cells. This effect is believed to bemediated by cells expressing the receptor that are present in the bonemarrow microenvironment. An agent that activates the receptor (such asparathyroid hormone—PTH), may therefore serve as a stimulant to enhancestem or progenitor cell production in vivo and in vitro. This representsan unexpected discovery with important clinical implications for thefield of progenitor cell transplantation.

Expanding the number of bone marrow derived progenitor cells is along-sought solution to the inadequate number of stem and progenitorcells available for transplantation in hematologic and oncologicdisease. A beneficial effect can be envisioned in at least the followingsettings: (i) the enhancement of stem and progenitor cell numbers invivo; this could be either prior to harvest to facilitate obtaining stemand progenitor cells, or to accelerate stem and progenitor cell recoveryfollowing bone marrow transplantation, and/or (ii) ex vivo expansion ofharvested stem and progenitor cells. A method to increase stem andprogenitor cell numbers in vivo would potentially reduce the time anddiscomfort associated with bone marrow/peripheral progenitor cellharvesting and increase the pool of progenitor cell donors. Currentlyapproximately 25% of autologous donor transplants are prohibited forlack of sufficient progenitor cells. In addition, less than 25% ofpatients in need of allogeneic transplant can find a histocompatibledonor. Umbilical cord blood banks currently exist and cover the broadracial make-up of the general population, but are currently restrictedin use to children due to inadequate progenitor cell numbers in thespecimens. A method to increase stem and progenitor cell numbers wouldpermit cord blood to be useful for adult patients, thereby expanding theuse of allogeneic transplantation.

It has also been discovered according to some aspects of the inventionthat enhancing the growth (including increase in self-renewal/numberincrease) or maintenance of hematopoietic stem or progenitor cellsthrough PTH/PTHrP stimulation will increase the ratio of normal toabnormal hematopoietic cells. A beneficial effect can be envisioned forleukemic and pre-leukemic conditions, where the progressive dominationof abnormal cells results in disease.

It is well known in the art that hematopoietic cells include pluripotentstem cells, multipotent progenitor cells (e.g., a lymphoid stem cell),and/or progenitor cells committed to specific hematopoietic lineages.The progenitor cells committed to specific hematopoietic lineages may beof T cell lineage, B cell lineage, dendritic cell lineage, Langerhanscell lineage and/or lymphoid tissue-specific macrophage cell lineage. Itis also known in the art that hematopoietic progenitor cells may or maynot include CD34⁺ cells. CD34⁺ cells are immature cells present in the“blood products” described below, express the CD34 cell surface marker,and are believed to include a subpopulation of cells with the“progenitor cell” properties defined above.

The hematopoietic stem and progenitor cells can be obtained from bloodproducts. A “blood product” as used in the present invention defines aproduct obtained from the body or an organ of the body containing cellsof hematopoietic origin. Such sources include unfractionated bonemarrow, umbilical cord, peripheral blood, liver, thymus, lymph andspleen. It will be apparent to those of ordinary skill in the art thatall of the aforementioned crude or unfractionated blood products can beenriched for cells having “hematopoietic progenitor cell”characteristics in a number of ways. For example, the blood product canbe depleted from the more differentiated progeny. The more mature,differentiated cells can be selected against, via cell surface moleculesthey express. Additionally, the blood product can be fractionatedselecting for CD34⁺ cells. As mentioned earlier, CD34⁺ cells are thoughtin the art to include a subpopulation of cells capable of self-renewaland pluripotentiality. Such selection can be accomplished using, forexample, commercially available magnetic anti-CD34 beads (Dynal, LakeSuccess, N.Y.). Unfractionated blood products can be obtained directlyfrom a donor or retrieved from cryopreservative storage.

Progeny of hematopoietic stem and progenitor cells comprise granulocytes(e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes(e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets), andmonocytes (e.g., monocytes, macrophages).

In important embodiments, the cell expressing a PTH/PTHrP receptor ispresent in the immediate vicinity of a hematopoietic stem or progenitorcell. In certain embodiments, the cell expressing a PTH/PTHrP receptoris a lymphoreticular stromal cell. “Lymphoreticular stromal cells” asused herein may include, but are not limited to, all cell types presentin a lymphoid tissue which are not lymphocytes or lymphocyte precursorsor progenitors, e.g., osteoblasts, epithelial cells, endothelial cells,mesothelial cells, dendritic cells, splenocytes and macrophages.Lymphoreticular stromal cells also include cells that would notordinarily function as lymphoreticular stromal cells, such asfibroblasts, which have been genetically altered to secrete or expresson their cell surface the factors necessary for the maintenance, growthand/or differentiation of hematopoietic stem and progenitor cells,including their progeny. Lymphoreticular stromal cells are derived fromthe disaggregation of a piece of lymphoid tissue (see discussion below).Such cells according to the invention are capable of supporting in vitrothe maintenance, growth and/or differentiation of hematopoietic stem andprogenitor cells, including their progeny. By “lymphoid tissue” it ismeant to include bone marrow, peripheral blood (including mobilizedperipheral blood), umbilical cord blood, placental blood, fetal liver,embryonic cells (including embryonic stem cells),aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue.“Lymphoid soft tissue” as used herein includes, but is not limited to,tissues such as thymus, spleen, liver, lymph node, skin, tonsil,adenoids and Peyer's patch, and combinations thereof.

Lymphoreticular stromal cells provide the supporting microenvironment inthe intact lymphoid tissue for the maintenance, growth and/ordifferentiation of hematopoietic progenitor cells, including theirprogeny. The microenvironment includes soluble and cell surface factorsexpressed by the various cell types which comprise the lymphoreticularstroma. Generally, the support which the lymphoreticular stromal cellsprovide may be characterized as both contact-dependent andnon-contact-dependent.

Lymphoreticular stromal cells may be autologous (“self”) ornon-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic)with respect to hematopoietic progenitor cells or antigen presentingcells. “Autologous,” as used herein, refers to cells from the samesubject. “Allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison. “Syngeneic,”as used herein, refers to cells of a different subject that aregenetically identical to the cell in comparison. “Xenogeneic,” as usedherein, refers to cells of a different species to the cell incomparison. Lymphoreticular stroma cells may be obtained from thelymphoid tissue of a human or a non-human subject at any time after theorgan/tissue has developed to a stage (i.e., the maturation stage) atwhich it can support the maintenance growth and/or differentiation ofhematopoietic stem and progenitor cells. The stage will vary betweenorgans/tissues and between subjects. In primates, for example, thematuration stage of thymic development is achieved during the secondtrimester. At this stage of development the thymus can produce peptidehormones such as thymulin, α₁ and β₄-thymosin, and thymopoietin, as wellas other factors required to provide the proper microenvironment for Tcell differentiation. The different maturation stages for the differentorgans/tissues and between different subjects are well known in the art.

Lymphoreticular stromal cells, preferably express the PTH/PTHrPreceptor. The lymphoid tissue from which lymphoreticular stromal cellsare derived usually determines the lineage-commitment hematopoietic stemand progenitor cells undertake, resulting in the lineage-specificity ofthe differentiated progeny. In certain embodiments, the lymphoreticularstromal cells are thymic stromal cells and the multipotent progenitorcells and/or committed progenitor cells are committed to a T celllineage. In other embodiments, the lymphoreticular stromal cells may besplenic stromal cells and the multipotent progenitor cells and/orcommitted progenitor cells are committed to a B cell lineage. Alsosurprising, according to the invention, has been the discovery that thehighest yield of differentiated progeny occurs when human hematopoieticprogenitor cells are cultured in the presence of xenogeneic (non-human)lymphoreticular stromal cells. Preferably the xenogeneic lymphoreticularstromal cells are of murine origin.

Various other embodiments are provided, wherein the lymphoreticularstromal cells may be genetically altered. In certain embodiments,lymphoreticular stromal cells, preferably express the PTH/PTHrP receptor(endogenously or via genetic alteration). The lymphoreticular stromalcells may be transfected with exogenous DNA that encodes, for example,one of the hematopoietic growth factors described elsewhere herein.

As mentioned earlier, lymphoreticular stromal cells are derived from thedisaggregation of a piece of lymphoid tissue, forming cell suspensions.Preferably, single cell suspensions are generated. These lymphoreticularstromal cell suspensions may be used directly, or made non-mitotic byprocedures standard in the tissue culture art. Examples of such methodsare irradiation of lymphoreticular stromal cells with a gamma-ray sourceor incubation of the cells with mitomycin C for a sufficient amount oftime to render the cells mitotically inactive. Mitotic inactivation ispreferred when the lymphoreticular stromal cells are of human origin (toeliminate progenitor cells that may be present in the suspension). Thelymphoreticular stromal cells may then be seeded into athree-dimensional matrix of the invention and permitted to attach to asurface of the porous, solid matrix. It should be noted that thelymphoreticular stromal cells may alternatively be cryopreserved forlater use or for storage and shipment to remote locations, such as foruse in connection with the sale of kits. Cryopreservation of cellscultured in vitro is well established in the art. Subsequent toisolation (and/or mitotic inactivation) of a cell sample, cells may becryopreserved by first suspending the cells in a cryopreservation mediumand then gradually freezing the cell suspension. Frozen cells aretypically stored in liquid nitrogen or at an equivalent temperature in amedium containing serum and a cryopreservative such as dimethylsulfoxide.

The co-culture of hematopoietic stem or progenitor cells (and progenythereof) with lymphoreticular stromal cells, according to certainaspects of the invention preferably occurs under conditions sufficientto produce a percent increase in the number of lymphoid tissue origincells deriving from the hematopoietic stem or progenitor cells. Theconditions used refer to a combination of conditions known in the art(e.g., temperature, CO₂ and O₂ content, nutritive media, time-length,etc.). The time sufficient to increase the number of cells is a timethat can be easily determined by a person skilled in the art, and canvary depending upon the original number of cells seeded. The amounts ofhematopoietic stem or progenitor cells and lymphoreticular stromal cellsinitially introduced (and subsequently seeded) into the porous solidmatrix may vary according to the needs of the experiment. The idealamounts can be easily determined by a person skilled in the art inaccordance with needs. Hematopoietic progenitor cells may be added atdifferent numbers. As an example, discoloration of the media over acertain period of time can be used as an indicator of confluency.Additionally, and more precisely, different numbers of hematopoieticstem and progenitor cells or volumes of the blood product can becultured under identical conditions, and cells can be harvested andcounted over regular time intervals, thus generating the “controlcurves”. These “control curves” can be used to estimate cell numbers insubsequent assays.

The conditions for determining colony forming potential are similarlydetermined. Colony forming potential is the ability of a cell to formprogeny. Assays for this are well known to those of ordinary skill inthe art and include seeding cells into a semi-solid matrix, treatingthem with growth factors, and counting the number of colonies.

In preferred embodiments of the invention, the hematopoietic stem andprogenitor cells may be harvested. “Harvesting” hematopoietic progenitorcells is defined as the dislodging or separation of cells from thematrix. This can be accomplished using a number of methods, such asenzymatic, non-enzymatic, centrifugal, electrical, or size-basedmethods, or preferably, by flushing the cells using media (e.g. media inwhich the cells are incubated). The cells can be further collected,separated, and further expanded generating even larger populations ofdifferentiated progeny.

As mentioned above, the stem and progenitor cells, and progeny thereof,can be genetically altered. Genetic alteration of a stem and progenitorcell includes all transient and stable changes of the cellular geneticmaterial which are created by the addition of exogenous geneticmaterial. Examples of genetic alterations include any gene therapyprocedure, such as introduction of a functional gene to replace amutated or nonexpressed gene, introduction of a vector that encodes adominant negative gene product, introduction of a vector engineered toexpress a ribozyme and introduction of a gene that encodes a therapeuticgene product. Natural genetic changes such as the spontaneousrearrangement of a T cell receptor gene without the introduction of anyagents are not included in this embodiment. Exogenous genetic materialincludes nucleic acids or oligonucleotides, either natural or synthetic,that are introduced into the stem and progenitor cells. The exogenousgenetic material may be a copy of that which is naturally present in thecells, or it may not be naturally found in the cells. It typically is atleast a portion of a naturally occurring gene which has been placedunder operable control of a promoter in a vector construct.

Various techniques may be employed for introducing nucleic acids intocells. Such techniques include transfection of nucleic acid-CaPO₄precipitates, transfection of nucleic acids associated with DEAE,transfection with a retrovirus including the nucleic acid of interest,liposome mediated transfection, and the like. For certain uses, it ispreferred to target the nucleic acid to particular cells. In suchinstances, a vehicle used for delivering a nucleic acid according to theinvention into a cell (e.g., a retrovirus, or other virus; a liposome)can have a targeting molecule attached thereto. For example, a moleculesuch as an antibody specific for a surface membrane protein on thetarget cell or a ligand for a receptor on the target cell can be boundto or incorporated within the nucleic acid delivery vehicle. Forexample, where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

In the present invention, the preferred method of introducing exogenousgenetic material into cells is by transducing the cells in situ on thematrix using replication-deficient retroviruses. Replication-deficientretroviruses are capable of directing synthesis of all virion proteins,but are incapable of making infectious particles. Accordingly, thesegenetically altered retroviral vectors have general utility forhigh-efficiency transduction of genes in cultured cells, and specificutility for use in the method of the present invention. Retroviruseshave been used extensively for transferring genetic material into cells.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell line with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with the viral particles) are provided in the art.

The major advantage of using retroviruses is that the viruses insertefficiently a single copy of the gene encoding the therapeutic agentinto the host cell genome, thereby permitting the exogenous geneticmaterial to be passed on to the progeny of the cell when it divides. Inaddition, gene promoter sequences in the LTR region have been reportedto enhance expression of an inserted coding sequence in a variety ofcell types. The major disadvantages of using a retrovirus expressionvector are (1) insertional mutagenesis, i.e., the insertion of thetherapeutic gene into an undesirable position in the target cell genomewhich, for example, leads to unregulated cell growth and (2) the needfor target cell proliferation in order for the therapeutic gene carriedby the vector to be integrated into the target genome. Despite theseapparent limitations, delivery of a therapeutically effective amount ofa therapeutic agent via a retrovirus can be efficacious if theefficiency of transduction is high and/or the number of target cellsavailable for transduction is high.

Yet another viral candidate useful as an expression vector fortransformation of cells is the adenovirus, a double-stranded DNA virus.Like the retrovirus, the adenovirus genome is adaptable for use as anexpression vector for gene transduction, i.e., by removing the geneticinformation that controls production of the virus itself. Because theadenovirus functions usually in an extrachromosomal fashion, therecombinant adenovirus does not have the theoretical problem ofinsertional mutagenesis. On the other hand, adenoviral transformation ofa target cell may not result in stable transduction. However, morerecently it has been reported that certain adenoviral sequences conferintrachromosomal integration specificity to carrier sequences, and thusresult in a stable transduction of the exogenous genetic material.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable vectors are available for transferring exogenous geneticmaterial into cells. The selection of an appropriate vector to deliver atherapeutic agent for a particular condition amenable to genereplacement therapy and the optimization of the conditions for insertionof the selected expression vector into the cell, are within the scope ofone of ordinary skill in the art without the need for undueexperimentation. The promoter characteristically has a specificnucleotide sequence necessary to initiate transcription. Optionally, theexogenous genetic material further includes additional sequences (i.e.,enhancers) required to obtain the desired gene transcription activity.For the purpose of this discussion an “enhancer” is simply anynontranslated DNA sequence which works contiguous with the codingsequence (in cis) to change the basal transcription level dictated bythe promoter. Preferably, the exogenous genetic material is introducedinto the cell genome immediately downstream from the promoter so thatthe promoter and coding sequence are operatively linked so as to permittranscription of the coding sequence. A preferred retroviral expressionvector includes an exogenous promoter element to control transcriptionof the inserted exogenous gene. Such exogenous promoters include bothconstitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a gene under the control of aconstitutive promoter is expressed under all conditions of cell growth.Exemplary constitutive promoters include the promoters for the followinggenes which encode certain constitutive or “housekeeping” functions:hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase(DHFR) (Scharfmann et al., 1991, Proc. Natl. Acad. Sci. USA,88:4626-4630), adenosine deaminase, phosphoglycerol kinase (PGK),pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al.,1989, Proc. Natl. Acad. Sci. USA, 86:10006-10010), and otherconstitutive promoters known to those of skill in the art. In addition,many viral promoters function constitutively in eukaryotic cells. Theseinclude: the early and late promoters of SV40; the long terminal repeats(LTRS) of Moloney Leukemia Virus and other retroviruses; and thethymidine kinase promoter of Herpes Simplex Virus, among many others.Accordingly, any of the above-referenced constitutive promoters can beused to control transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressedonly or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). Induciblepromoters include responsive elements (REs) which stimulatetranscription when their inducing factors are bound. For example, thereare REs for serum factors, steroid hormones, retinoic acid and cyclicAMP. Promoters containing a particular RE can be chosen in order toobtain an inducible response and in some cases, the RE itself may beattached to a different promoter, thereby conferring inducibility to therecombinant gene. Thus, by selecting the appropriate promoter(constitutive versus inducible; strong versus weak), it is possible tocontrol both the existence and level of expression of a therapeuticagent in the genetically modified cell. Selection and optimization ofthese factors for delivery of a therapeutically effective dose of aparticular therapeutic agent is deemed to be within the scope of one ofordinary skill in the art without undue experimentation, taking intoaccount the above-disclosed factors and the clinical profile of thepatient.

In addition to at least one promoter and at least one heterologousnucleic acid encoding the therapeutic agent, the expression vectorpreferably includes a selection gene, for example, a neomycin resistancegene, for facilitating selection of cells that have been transfected ortransduced with the expression vector. Alternatively, the cells aretransfected with two or more expression vectors, at least one vectorcontaining the gene(s) encoding the therapeutic agent(s), the othervector containing a selection gene. The selection of a suitablepromoter, enhancer, selection gene and/or signal sequence (describedbelow) is deemed to be within the scope of one of ordinary skill in theart without undue experimentation.

The selection and optimization of a particular expression vector forexpressing a specific gene product in an isolated cell is accomplishedby obtaining the gene, preferably with one or more appropriate controlregions (e.g., promoter, insertion sequence); preparing a vectorconstruct comprising the vector into which is inserted the gene;transfecting or transducing cultured cells in vitro with the vectorconstruct; and determining whether the gene product is present in thecultured cells.

TABLE 1 Human Gene Therapy Protocols Approved by RAC: 1990-1994 Severecombined Autologous lymphocytes transduced with human Jul. 31, 1990immune deficiency ADA gene (SCID) due to ADA deficiency Advanced cancerTumor-infiltrating lymphocytes transduced with tumor Jul. 31, 1990necrosis factor gene Advanced cancer Immunization with autologous cancercells transduced Oct. 07, 1991 with tumor necrosis factor gene Advancedcancer Immunization with autologous cancer cells transduced Oct. 07,1991 with interleukin-2 gene Asymptomatic patients Murine Retro viralvector encoding HIV-1 genes Jun. 07, 1993 infected with HIV-1[HIV-IT(V)] AIDS Effects of a transdominant form of rev gene on AIDSJun. 07, 1993 Intervention Advanced cancer Human multiple-drugresistance (MDR) gene transfer Jun. 08, 1993 HIV infection Autologouslymphocytes transduced with catalytic Sep. 10, 1993 ribozyme thatcleaves HIV-1 RNA (Phase I study) Metastatic melanoma Geneticallyengineered autologous tumor vaccines Sep. 10, 1993 producinginterleukin-2 HIV infection Murine Retro viral vector encoding HIV-IT(V)genes Dec. 03, 1993 (open label Phase I/II trial) HIV infection Adoptivetransfer of syngeneic cytotoxic T lymphocytes Mar. 03, 1994 (identicaltwins) (Phase I/II pilot study) Breast cancer (chemo- Use of modifiedRetro virus to introduce chemotherapy Jun. 09, 1994 protection duringresistance sequences into normal hematopoietic cells therapy) (pilotstudy) Fanconi's anemia Retro viral mediated gene transfer of theFanconi anemia Jun. 09, 1994 complementation group C gene tohematopoietic progenitors Metastatic prostate Autologous humangranulocyte macrophage-colony ORDA/NIH Carcinoma stimulating factor genetransduced prostate cancer vaccine Aug. 03, 1994* *(first protocol to beapproved under the accelerated review process; ORDA = Office ofRecombinant DNA Activities) Metastatic breast cancer In vivo infectionwith breast-targeted Retro viral vector Sep. 12, 1994 expressingantisense c-fox or antisense c-myc RNA Metastatic breast cancerNon-viral system (liposome-based) for delivering human Sep. 12, 1994(refractory or recurrent) interleukin-2 gene into autologous tumor cells(pilot study) Mild Hunter syndrome Retro viral-mediated transfer of theiduronate-2-sulfatase Sep. 13, 1994 gene into lymphocytes Advancedmesothelioma Use of recombinant adenovirus (Phase I study) Sep. 13, 1994

The foregoing (Table 1), represent only examples of genes that can bedelivered according to the methods of the invention. Suitable promoters,enhancers, vectors, etc., for such genes are published in the literatureassociated with the foregoing trials. In general, useful genes replaceor supplement function, including genes encoding missing enzymes such asadenosine deaminase (ADA) which has been used in clinical trials totreat ADA deficiency and cofactors such as insulin and coagulationfactor VIII. Genes which affect regulation can also be administered,alone or in combination with a gene supplementing or replacing aspecific function. For example, a gene encoding a protein whichsuppresses expression of a particular protein-encoding gene can beadministered. The invention is particularly useful in delivering geneswhich stimulate the immune response, including genes encoding viralantigens, tumor antigens, cytokines (e.g. tumor necrosis factor) andinducers of cytokines (e.g. endotoxin).

Employing the culture conditions described in greater detail below, itis possible according to the invention to preserve hematopoietic stemand progenitor cells and to stimulate the expansion of hematopoieticstem and progenitor cell number and/or colony forming unit potential.Once expanded, the cells, for example, can be returned to the body tosupplement, replenish, etc. a patient's hematopoietic stem andprogenitor cell population. This might be appropriate, for example,after an individual has undergone chemotherapy. There are certaingenetic conditions wherein hematopoietic stem and progenitor cellnumbers are decreased, and the methods of the invention may be used inthese situations as well.

It also is possible to take the increased numbers of hematopoietic stemand progenitor cells produced according to the invention and stimulatethem with hematopoietic growth agents that promote hematopoietic cellmaintenance, expansion and/or differentiation, and also influence celllocalization, to yield the more mature blood cells, in vitro. Suchexpanded populations of blood cells may be applied in vivo as describedabove, or may be used experimentally as will be recognized by those ofordinary skill in the art. Such differentiated cells include thosedescribed above, as well as T cells, plasma cells, erythrocytes,megakaryocytes, basophils, polymorphonuclear leukocytes, monocytes,macrophages, eosinophils and platelets.

In all of the in vitro and ex vivo culturing methods according to theinvention, except as otherwise provided, the media used is that which isconventional for culturing cells. Examples include RPMI, DMEM, Iscove's,etc. Typically these media are supplemented with human or animal plasmaor serum. Such plasma or serum can contain small amounts ofhematopoietic growth factors. The media used according to the presentinvention, however, can depart from that used conventionally in theprior art.

The growth agents of particular interest in connection with the presentinvention are hematopoietic growth factors. By hematopoietic growthfactors, it is meant factors that influence the survival, proliferationor differentiation of hematopoietic stem and progenitor cells. Growthagents that affect only survival and proliferation, but are not believedto promote differentiation, include the interleukins 3, 6 and 11, stemcell factor and FLT-3 ligand. Hematopoietic growth factors that promotedifferentiation include the colony stimulating factors such as GMCSF,GCSF, MCSF, Tpo, Epo, Oncostatin M, and interleukins other than IL-3, 6and 11. The foregoing factors are well known to those of ordinary skillin the art. Most are commercially available. They can be obtained bypurification, by recombinant methodologies or can be derived orsynthesized synthetically.

“Stromal cell conditioned medium” refers to medium in which theaforementioned lymphoreticular stromal cells have been incubated. Theincubation is performed for a period sufficient to allow the stromalcells to secrete factors into the medium. Such “stromal cell conditionedmedium” can then be used to supplement the culture of hematopoietic stemand progenitor cells promoting their proliferation and/ordifferentiation.

Thus, when cells are cultured without any of the foregoing agents, it ismeant herein that the cells are cultured without the addition of suchagent except as may be present in serum, ordinary nutritive media orwithin the blood product isolate, unfractionated or fractionated, whichcontains the hematopoietic stem and progenitor cells.

One method for modulating hematopoietic cell function according to theinvention is a method for enhancing mobilization of hematopoieticprogenitor cells by using agents that activate the PTH/PTHrP receptor.Current practice during bone marrow transplantation involves theisolation of bone marrow cells from the bone marrow and/or peripheralblood of donor subjects. About one third of these subjects do not“yield” enough hematopoietic progenitor cells from their bone marrowand/or peripheral blood so that their marrow can be considered suitablefor transplantation. Using the methods of the invention, the “yield” maybe enhanced. For example, agents that activate the PTH/PTHrP receptorcould result in “mobilization” of hematopoietic progenitor cells and theefficiency of hematopoietic progenitor cell isolation from subjectstreated with such agents may be improved (especially from the subject'speripheral blood). This then results in an increase in the number ofdonor samples that may be used in transplantation.

Thus, in some aspects a method for enhancing mobilization ofhematopoietic cells in a subject is provided. The method involvesadministering to a subject an agent that activates the PTH/PTHrPreceptor to enhance mobilization of hematopoietic progenitor cells inthe subject.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. Human hematopoietic progenitorcells and human subjects are particularly important embodiments.

As used herein a “an agent that activates the PTH/PTHrP receptor” is acompound that includes Parathyroid hormone (PTH), parathyroidrelated-protein (PTHrP), and analogues thereof.

The term “obtaining” as in “obtaining the agent that activates thePTH/PTHrP receptor” is intended to include purchasing, synthesizing orotherwise acquiring the agent (or indicated substance or material).

The normal function of PTH is to maintain extracellular fluid calciumconcentration. PTH acts directly on bone and kidney and indirectly onthe intestines. PTH production in healthy individuals is closelyregulated by the concentration of serum ionized calcium. Tendenciestowards hypocalcemia, for example, induced by a calcium-deficient diet,are balanced by an increased PTH secretion. The increase in PTH levelsincreases the rate of bone resorption, thereby increasing the calciumflow from bone into blood, reduces the renal clearance of calcium, andincreases the efficiency of calcium absorption in the intestines.

The physiological role of the parathyroid hormone-related protein(PTHrP) is not fully understood, but is thought to be acting principallyas a paracrine or autocrine factor. PTHrP plays a role in fetaldevelopment as well as in adult physiology. PTHrP is produced by manycell types, including brain, pancreas, heart, lung, memory tissue,placenta, endothelial, and smooth muscle cells. In adults, PTHrP isthought to have little to do with calcium homeostasis, except in diseasestates.

PTH and PTHrP are distinct proteins and products of different genes.However, they share a similar bioactivity profile and a very limitedsequence homology, indicating that they may have evolved from a commonancestral gene. Eight out of the 13 first amino acid residues at theN-terminus are identical. Both PTH, an 84 amino acid residues peptide,and PTHrP, a 139 to 173 amino acid residues peptide, bind to the PTHreceptor (often referred to as the PTH/PTHrP receptor) and stimulate thesame intracellular signaling pathways.

Parathyroid hormone (PTH) is an 84 amino acid polypeptide which isnormally secreted from the parathyroid glands. PTH has an importantphysiological role to maintain serum calcium within a narrow range.Furthermore, it has anabolic properties when given intermittently. Thishas been well documented in a number of animal and open clinicalstudies, recently reviewed by Dempster, D. W. et al. (Endocrine Reviews1993, vol. 14, 690-709). PTH has a multitude of effects on bone. Part ofit is through the remodeling cycle. PTH causes both increased activationfrequency and a positive balance per cycle. Human PTH may be obtainedthrough peptide synthesis or from genetically engineered yeast,bacterial or mammalian cell hosts. Synthetic human PTH is commerciallyavailable from Bachem Inc., Bubendorf, Switzerland. Production ofrecombinant human parathyroid hormone is disclosed in e.g. EP-B-0383751.

The mature circulating form of parathyroid hormone is comprised of 84amino acid residues. For most bone-related activities the truncated formof PTH, PTH(1-34), is a full agonist like the native 84 amino-acidhormone. Amino-terminal truncation results in polypeptides that arecompetitive antagonists of PTH-stimulated adenylate cyclase. Forexample, [Tyr³⁴]bPTH(7-34)NH₂ retains moderate affinity for renal PTHreceptors, but does not have any agonist activity; weak receptor bindingactivity is retained in a fragment as small as PTH(25-34) (M.Rosenblatt, et al., 1980, Endocrinol., 107:545-550). In contrast,carboxyl-terminal truncations of PTH(1-34) produce agonists withprogressively lower affinities. PTH(1-25) is inactive, however, it ispossible to construct mutants of PTH(1-25) that will have activity(Shimizu et al. J. Biol. Chem. 276:52 (2001)). The principalreceptor-binding domain of PTH is reported to include amino acidresidues 25-34 and the principal activation domain is reported toinclude amino acid residues 1-6.

The term “parathyroid hormone” (PTH) encompasses naturally occurringhuman PTH, as well as synthetic or recombinant PTH (rPTH). Further, theterm “parathyroid hormone” encompasses full-length PTH(1-84) as well asPTH fragments. It will thus be understood that fragments of PTHvariants, in amounts giving equivalent biological activity to PTH(1-84),can be incorporated in the formulations according to the invention, ifdesired. In this context, the term “biologically active” should beunderstood as eliciting a sufficient response in a bioassay for PTHactivity according to the methods described herein. Fragments of PTHincorporate at least the amino acid residues of PTH necessary for abiological activity similar to that of intact PTH. Examples of suchfragments are PTH(1-31), PTH(1-34), PTH(1-36), PTH(1-37), PTH(1-38),PTH(1-41), PTH(28-48), PTH(1-25) variants and PTH(25-39).

The term “parathyroid hormone” also encompasses variants and functionalanalogs of PTH. The present invention thus includes pharmaceuticalformulations comprising such PTH variants and functional analogues,carrying modifications like substitutions, deletions, insertions,inversions or cyclisations, but nevertheless having substantially thebiological activities of parathyroid hormone. Stability-enhancedvariants of PTH are known in the art from e.g. WO 92/11286 and WO93/20203. Variants of PTH can e.g. incorporate amino acid substitutionsthat improve PTH stability and half-life, such as the replacement ofmethionine residues at positions 8 and/or 18, and replacement ofasparagine at position 16.

Biologically active PTH/PTHrP analogs of any mammalian species, e.g.,human, bovine, porcine, or rabbit, can be used in the methods of thepresent inventions, with human analogues being preferred. SuitablePTH/PTHrP analogs for use in accordance with the present inventioninclude those described in U.S. Pat. Nos. 5,589,452, 5,849,695,5,695,955, 6,362,163, 6,147,186 and 6,583,114. Cyclized PTH analogs aredisclosed in e.g. WO 98/05683.

U.S. Pat. Nos. 5,589,452, 5,695,955, and 6,583,114 describe syntheticPTH analogs of PTH and PTHrP in which certain amino acid residues(22-31) form an amphipathic alpha helix.

U.S. Pat. No. 5,849,695 describes PTH analogs of PTH and PTHrP in whichthe serine amino acid at position 3, the glutamine amino acid atposition 6, the histidine amino acid at position 9 or combinationsthereof are substituted by other natural or synthetic amino acids.

U.S. Pat. Nos. 6,362,163 and 6,147,186 describe PTHrP analogs that havebeen converted into PTH-2 receptor agonists by the substitution of oneor more amino acid residues of PTHrP to the corresponding residue(s) ofPTH (e.g., the amino acid sequence is altered at amino acid residues 5and 23, for example, (Ile₅, Trp₂₃) PTHrP-(1-36) wherein the alterationat PTHrP amino acid residue 5 is an amino acid substitution of histidinefor isoleucine, and the alteration at PTHrP amino acid residue 23 is anamino acid substitution of phenylalanine for tryptophan).

Various PTH/PTHrP products, including fragments, variants and analogues,are already commercially available, or in various stages of development.For example, synthetic bovine PTH(1-34) is available from Bachem, Inc.,Torrance, Calif.; synthetic human PTHrP(1-34) amide is available fromMerck Sharp and Dohme, West Point, Pa.; BIM-44058, a PTH (1-34) analog,is manufactured by Ipseni Ltd, Slough, Berkshire, U.K.; the PTH analogueOstabolin-C™ is manufactured by Zelos Therapeutics Inc., Ottawa, ON,Canada; and the recombinant PTH analogue Forteo™ is manufactured by EliLilly and Company, Indianapolis, Ind.

The Ostabolin-C™ peptide is a 31 amino acid peptide derivative of PTH.The Ostabolin-C™ peptide differs from PTH in that the peptide has beencyclized by a lactam moiety between Glu²² and Lys²⁶ and replacement ofLys²⁷ with Leu. The Ostabolin-C™ peptide can be represented byLeu²⁷cyclo[Glu²²-Lys²⁶]-hPTH(1-31)-NH₂ as shown in FIG. 8 (SEQ ID NO:1).

Cyclized PTH analogues are also described in U.S. Pat. Nos. 5,556,940;5,955,425; 6,110,892; 6,316,410; and 6,541,450 the teachings of all ofwhich are hereby incorporated by reference in their entirety.

Under some circumstances, PTH is a bone anabolic agent, and promotesbone formation. However, PTH can stimulate bone resorption as well. Ithas been reported that high-dose, continuous administration of PTHresults in a lowered bone mass but low-dose, intermittent administrationof PTH can increase bone mass. PTH administered continuously reportedlycauses an increase in the number of bone cells, including osteoclasts,and an increase in bone remodeling. These increases reportedly areapparent within hours after PTH administration and persist for hoursafter PTH is withdrawn. PTH administration intermittently over days inhumans and animals reportedly leads to a net stimulation of boneformation. For example, see Neer et al., 2001, N. Engl. J. Med.,344:1434-1441. In contrast, continuous exposure to elevated levels ofPTH leads to osteoclast-mediated bone resorption. Several groups haveinvestigated the use of PTH and PTHrP analogues as agents to treatosteoporosis. These efforts are described in U.S. Pat. No. 5,747,456;U.S. Pat. No. 5,849,695; U.S. Pat. No. 4,656,250; U.S. Pat. No.6,051,686; and U.S. Pat. No. 6,316,410.

In one embodiment the subject is a bone marrow donor. By enhancingmobilization of bone marrow cells, the need for bone marrow isolationmay be obviated. As a result of this mobilization, bone marrow cellsleave the bone marrow and enter the blood circulation of the subjectundergoing treatment. The circulating bone marrow cells can then beeasily isolated using the techniques of the invention or other methodsknow in the art. For instance, these methods may reduce the need forlarge bone marrow donations for therapeutic procedures. The methodsenable the isolation of hematopoietic stem and progenitor cells fromperipheral blood by encouraging localization from the bone marrow to theblood and thus, eliminating the need for bone marrow donation.

One of skill in the art would be aware of methods for isolatinghematopoietic stem and progenitor cells from peripheral blood. Forexample blood in PBS is loaded into a tube of Ficoll (Ficoll-Paque,Amersham) and centrifuged at 1500 rpm for 25-30 minutes. Aftercentrifugation the white center ring is collected as containinghematopoietic stem cells.

Hematopoietic stem and progenitor cell manipulation is also useful as asupplemental treatment to chemotherapy, e.g., hematopoietic progenitorcells may be caused to localize into the peripheral blood and thenisolated from a subject that will undergo chemotherapy, and after thetherapy the cells can be returned (e.g. ex vivo therapy may also beperformed on the isolated cells). Thus, the subject in some embodimentsis a subject undergoing or expecting to undergo an immune cell depletingtreatment such as chemotherapy. Most chemotherapy agents used act bykilling all cells going through cell division. Bone marrow is one of themost prolific tissues in the body and is therefore often the organ thatis initially damaged by chemotherapy drugs. The result is that bloodcell production is rapidly destroyed during chemotherapy treatment, andchemotherapy must be terminated to allow the hematopoietic system toreplenish the blood cell supplies before a patient is re-treated withchemotherapy. This can be avoided using the methods of the invention.

Once the hematopoietic stem and progenitor cells are mobilized from thebone marrow to the peripheral blood, a blood sample can be isolated inorder to obtain the hematopoietic progenitor cells. These cells can betransplanted immediately or they can be processed in vitro first. Forinstance, the cells can be expanded in vitro and/or they can besubjected to an isolation or enrichment procedure. It will be apparentto those of ordinary skill in the art that the crude or unfractionatedblood products can be enriched for cells having “hematopoieticprogenitor cell” characteristics. Some of the ways to enrich include,e.g., depleting the blood product from the more differentiated progeny.The more mature, differentiated cells can be selected against, via cellsurface molecules they express. Additionally, the blood product can befractionated selecting for CD34⁺ cells. Such selection can beaccomplished using, for example, commercially available magneticanti-CD34 beads (Dynal, Lake Success, N.Y.). In preferred embodiments,however, the methods of the invention may be used to isolate thehematopoietic stem and progenitor cells.

Methods for isolation of hematopoietic stem and progenitor cells arewell-known in the art, and typically involve purification techniquesbased on cell surface markers and functional characteristics. Thehematopoietic stem and progenitor cells can be isolated from bonemarrow, blood, cord blood, fetal liver and yolk sac, and give rise tomultiple hematopoietic lineages and can reinitiate hematopoiesis for thelife of a recipient. (See Fei, R., et al., U.S. Pat. No. 5,635,387;McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat.No. 5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, etal., U.S. Pat. No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599;Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al. 1996.) Whentransplanted into lethally irradiated animals or humans, hematopoieticstem cells can repopulate the erythroid, neutrophil-macrophage,megakaryocyte and lymphoid hematopoietic cell pool. In vitro,hematopoietic stem cells can be induced to undergo at least someself-renewing cell divisions or can be induced to differentiate to thesame lineages observed in vivo. Accordingly, methods of the inventioncan involve the in vitro expansion of hematopoietic stem and progenitorcells by way of co-culture with stimulated PTH/PTHrP receptor expressingcells, thereby recapitulating the in vivo microenvironment.

Hematopoietic stems for use with co-culture-based methods of theinvention can be obtained from pluripotent stem cell sources as well.For example, U.S. Pat. No. 5,914,268 describes a pluripotent cellpopulation for use in the development into hematopoietic cells,progenitors and progeny thereof. The pluripotent cell population isderived by culturing an embryonic stem cell population to obtain anembryoid body cell population, which is then followed by culturing saidembryoid body cell population under conditions effective to produce saidpluripotent cell population. The culturing conditions comprise anembryonic blast cell medium.

The invention further provides methods of immunizing against and/ortreating a disorder or disease, such as for example an infectiousdisease, in an individual. The methods generally involve administeringto a subject the compounds of the invention in an amount effective tostimulate hematopoiesis.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, e.g., causing regression of the disease,e.g., to completely or partially remove symptoms of the disease.

The methods of the invention can be used to treat any disease ordisorder in which it is desirable to increase the production ofhematopoietic stem and progenitor cells, support the maintenance orsurvival of hematopoietic stem and progenitor cells, or mobilizehematopoietic stem cells. For example, the methods of the invention canbe used to treat patients requiring a bone marrow transplant or ahematopoietic stem or progenitor cell transplant, such as cancerpatients undergoing chemo and/or radiation therapy. Methods of thepresent invention are particularly useful in the treatment of patientsundergoing chemotherapy or radiation therapy for cancer, includingpatients suffering from myeloma, non-Hodgkin's lymphoma, Hodgkinslyphoma, or leukaemia.

Treatment can be used as a means to increase the amount of hematopoieticstem and progenitor cells in disorders where the progressive dominationof abnormal hematopoietic cells results in disease, such as in disordersof chronic leukemia (e.g., chronic myeloid, chronic myelogenous orchronic granulocytic leukemia), acute leukemia (e.g., acutelymphoblastic leukemia or acute nonlymphoblastic leukemia) andpre-leukemia (e.g., myelodysplasia). Abnormal cells that can beeffectively reduced or eradicated include leukemic cells, such aslymphoblastic leukemic cells. Treatment enables an increase in the ratioof normal to abnormal hematopoietic cells, thereby changing thephenotype of the malignancy such that it is ameliorated or eradicated.

Treatment can further be used as a means to increase the amount ofmature cells derived from hematopoietic stem cells (e.g., erythrocytes).For example, disorders or diseases characterized by a lack of bloodcells, or a defect in blood cells, can be treated by increasing theproduction of hematopoietic stem cells. Such conditions includethrombocytopenia (platelet deficiency), and anemias such as aplasticanemia, sickle cell anemia, fanconi's anemia, and acute lymphocyticanemia.

Disorders treated by methods of the invention can be the result of anundesired side effect or complication of another primary treatment, suchas radiation therapy, chemotherapy, or treatment with a bone marrowsuppressive drug, such as zidovadine, chloramphenical or gangciclovir.Such disorders include neutropenias, anemias, thrombocytopenia, andimmune dysfunction. In addition, methods of the invention can be used totreat damage to the bone marrow caused by unintentional exposure totoxic agents or radiation.

The disorder to be treated can also be the result of an infection (e.g.,viral infection, bacterial infection or fungal infection) causing damageto stem or progenitor cells.

Immunodeficiencies, such as T and/or B lymphocytes deficiencies, orother immune disorders, such as rheumatoid arthritis and lupus, can alsobe treated according to the methods of the invention. Suchimmunodeficiencies may also be the result of an infection (for exampleinfection with HIV leading to AIDS), or exposure to radiation,chemotherapy or toxins.

In addition to the above, further conditions which can benefit fromtreatment using methods of the invention include, but are not limitedto, lymphocytopenia, lymphorrhea, lymphostasis, erythrocytopenia,erthrodegenerative disorders, erythroblastopenia, leukoerythroblastosis;erythroclasis, thalassemia, myelo fibrosis, thrombocytopenia,disseminated intravascular coagulation (DIC), immune (autoimmune)thrombocytopenic purpura (ITP), HIV inducted ITP, myelodysplasia;thrombocytotic disease, thrombocytosis, congenital neutropenias (such asKostmann's syndrome and Schwachman-Diamond syndrome), neoplasticassociated—neutropenias, childhood and adult cyclic neutropaenia;post-infective neutropaenia; myelo-dysplastic syndrome; and neutropaeniaassociated with chemotherapy and radiotherapy.

Also benefiting from treatment according to methods of the invention areindividuals who are healthy, but who are at risk of being affected byany of the diseases or disorders described herein (“at-risk”individuals). At-risk individuals include, but are not limited to,individuals who have a greater likelihood than the general population ofbecoming cytopenic or immune deficient. Individuals at risk for becomingimmune deficient include, but are not limited to, individuals at riskfor HIV infection due to sexual activity with HIV-infected individuals;intravenous drug users; individuals who may have been exposed toHIV-infected blood, blood products, or other HIV-contaminated bodyfluids; babies who are being nursed by HIV-infected mothers; individualswho were previously treated for cancer, e.g., by chemotherapy orradiotherapy, and who are being monitored for recurrence of the cancerfor which they were previously treated; and individuals who haveundergone bone marrow transplantation or any other organtransplantation, or patients anticipated to undergo chemotherapy orradiation therapy or be a donor of stem cells for transplantation.

A reduced level of immune function compared to a normal subject canresult from a variety of disorders, diseases infections or conditions,including immunosuppressed conditions due to leukemia, renal failure;autoimmune disorders, including, but not limited to, systemic lupuserythematosus, rheumatoid arthritis, auto-immune thyroiditis,scleroderma, inflammatory bowel disease; various cancers and tumors;viral infections, including, but not limited to, human immunodeficiencyvirus (HIV); bacterial infections; and parasitic infections.

A reduced level of immune function compared to a normal subject can alsoresult from an immunodeficiency disease or disorder of genetic origin,or due to aging. Examples of these are immunodeficiency diseasesassociated with aging and those of genetic origin, including, but notlimited to, hyperimmunoglobulin M syndrome, CD40 ligand deficiency, IL-2receptor deficiency, γ-chain deficiency, common variableimmunodeficiency, Chediak-Higashi syndrome, and Wiskott-Aldrichsyndrome.

A reduced level of immune function compared to a normal subject can alsoresult from treatment with specific pharmacological agents, including,but not limited to chemotherapeutic agents to treat cancer; certainimmunotherapeutic agents; radiation therapy; immunosuppressive agentsused in conjunction with bone marrow transplantation; andimmunosuppressive agents used in conjunction with organ transplantation.

An “immune system deficiency” shall mean a disease or disorder in whichit would be useful to boost a subject's immune response for example toeliminate a tumor or cancer (e.g., tumors of the brain, lung (e.g.,small cell and non-small cells), ovary, breast, prostate, colon, as wellas other carcinomas and sarcomas) or an infection in a subject.

The compounds of the invention may be administered to the subject aloneor in combination with an antigen, such as a tumor antigen, a viral,bacterial, or fungal antigen or other therapeutic.

Examples of infectious virus include: Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1, also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fevervirus); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxyiridae (variola virsues, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatitides (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1—internally transmitted; class 2—parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.tuberculosis, M. avium, M. Intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasturellamulticoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomeyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

When the cells or any compounds of the invention (referred to astherapeutic compositions), such as PTH are administered to a subject,the therapeutic compositions may be administered in pharmaceuticallyacceptable preparations. Such preparations may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents.

The therapeutic composition may be administered by any conventionalroute, including injection or by gradual infusion over time. Theadministration may, depending on the composition being administered, forexample, be oral, pulmonary, intravenous, intraperitoneal,intramuscular, intracavity, subcutaneous, nasal or transdermal.Techniques for preparing aerosol delivery systems containing activeagents are well known to those of skill in the art. Generally, suchsystems should utilize components which will not significantly impairthe biological properties of the active agents (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing aerosols without resort to undueexperimentation. When using antisense preparations, intravenous or oraladministration are preferred. The compositions are administered ineffective amounts. An “effective amount” is that amount of a compositionthat alone, or together with further doses, produces the desiredresponse, e.g. results in an increase in hematopoietic progenitor cellsin the bone marrow. The term “therapeutic composition” is usedsynonymously with the terms “active compound”, “active agent” or “activecomposition” and as used herein refers to any of the active compounds ofthe invention which produce a biological effect, e.g., PTH, PTHanalogues such as those disclosed in U.S. Pat. Nos. 4,086,196,6,541,450, and WO93/06845, incorporated herein by reference, enrichedhematopoietic stem cell preparations, etc. In the case of treating aparticular disease or condition characterized by immune deficiency, thedesired response is any improvement in immune system function. This mayinvolve only an increase in the actual numbers of hematopoietic stemcells, slowing of onset or progression of an infectious disease arisingfrom the immune system dysfunction, temporarily, although morepreferably, it involves an actual improvement in the prevention ofdisease permanently. This can be monitored by routine methods.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of therapeutic compositionfor producing the desired response in a unit of weight or volumesuitable for administration to a patient. The response can, for example,be measured by determining the effect on cell mobilization followingadministration of the therapeutic composition via a reporter system, orby isolating cells and measuring mobility in vitro. Other assays will beknown to one of ordinary skill in the art and can be employed formeasuring the level of the response.

When administered, pharmaceutical preparations of the invention areapplied in pharmaceutically acceptable amounts and in pharmaceuticallyacceptable compositions. Such preparations may routinely contain salts,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic ingredients. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic,naphthalene-2-sulfonic, pamoic, 3-hydroxy-2-naphthalenecarboxylic, andbenzene sulfonic. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,ammonium, magnesium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and salts thereof (1-2%W/V); citric acid and salts thereof (1-3% W/V); boric acid and saltsthereof (0.5-2.5% W/V); and phosphoric acid and salts thereof (0.8-2%W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal(0.004-0.02% W/V).

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular combination oftherapeutic agents selected, the severity of the condition or disorderbeing treated, or prevented, the condition of the patient, and thedosage required for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, transdermal, sublingual or intramuscular, infusion, parenteral,intravenous, intramuscular, intracavity, as a feed additive, as anaerosol, buccal, aural (e.g., via eardrops), intranasal, inhalation, orsubcutaneous. Direct injection could also be preferred for localdelivery to the site of injury.

Doses of PTH and/or PTHrP administered can be between about 5 to about100 micrograms, between about 5 to about 150 micrograms, at least about5 micrograms, at least about 10 micrograms, at least about 20micrograms, at least about 25 micrograms, at least about 40 micrograms,at least about 50 micrograms, at least about 60 micrograms, at leastabout 75 micrograms, at least about 100 micrograms and at least about150 micrograms per dose.

Although at present subcutaneous administration is routinely employed inthe administration of PTH and/or PTHrP, oral administration may bepreferred for treatment because of the convenience of the subject(patient) as well as the dosing schedule. Generally, daily oral doses ofactive compounds will be from about 0.1 microgram per day to 1000micrograms per day. It is expected that oral doses in the range of 0.5to 500 micrograms, in one or several administrations per day, will yieldthe desired results.

Where PTH (1-34) is to be administered it is preferred that a singledaily dose in the range of about 10 to about 250 micrograms per day isadministered. Even more preferably, a single daily dose in the rangeabout 40 to about 100 micrograms per day is administered. Morepreferably still, a single daily dose of about 100 micrograms should beadministered. Where PTH (1-84) is to be administered it is preferredthat a single daily dose in the range of about 10 to about 250micrograms per day is administered. Even more preferably, a single dailydose in the range about 120 to about 170 micrograms per day isadministered. More preferably still, a single daily dose of around about120 micrograms should be administered.

The exact dosages used may be adjusted appropriately to achieve desireddrug levels, local or systemic, depending upon the particular propertiesof the PTH molecule administered, including its molecular weight andstability, and the mode of administration. For example, it is expectedthat intravenous administration would be from an order to several ordersof magnitude lower dose per day compared to the oral doses. In the eventthat the response in a subject is insufficient at such doses, evenhigher doses (or effective higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits.

Preferably the polypeptides of the invention are administeredintermittently, which is known in the art to promote anabolic efficacyof PTH, PTHrP and its analogues. Preferred intermittent administrationschedules include daily, every second day, every third day, twice perweek, every fourth day, every fifth day, every sixth day, and once perweek.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds of the inventioninto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compounds of the invention into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the compounds of theinvention. This preparation is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using those suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptabledilutant or solvent, for example as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono or di-glycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables. Carrierformulations suitable for oral, subcutaneous, intravenous,intramuscular, etc. are well known in the art.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, syrups, elixirs orlozenges, each containing a predetermined amount of the compounds of theinvention. Compositions suitable for any pulmonary delivery typicallyare formulated and/or are contained in a nebulizer.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds of the invention, increasingconvenience to the subject and the physician, yet are constructed toprovide the anabolic benefit of the polypeptides of the invention. Manytypes of release delivery systems are available and known to those ofordinary skill in the art. They include polymer based systems such aspolylactic and polyglycolic acid, polyanhydrides and polycaprolactone,nonpolymer systems that are lipids including sterols such ascholesterol, liposomes; phospholipids; hydrogel release systems;silastic systems; peptide based system; implants and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe polypeptide is contained in a form within a matrix, found in U.S.Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and5,407,686. In addition, pump-based delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions.

“Long-term” release, as used herein, means that the implant isconstructed and arranged to deliver therapeutic levels of the activeingredient for at least 7 days, preferably for 30-60 days and morepreferably for longer periods of time (e.g., 12 months or longer). Theimplant may be positioned at a site of injury, but need not be.Long-term sustained release implants are well-known to those of ordinaryskill in the art and include some of the release systems describedabove. One such implant system is described in U.S. Pat. No. 6,159,490.

Other protocols for the administration of therapeutic compositions willbe known to one of ordinary skill in the art, in which the dose amount,schedule of injections, sites of injections, mode of administration andthe like vary from the foregoing. Administration of therapeuticcompositions to mammals other than humans, e.g. for testing purposes orveterinary therapeutic purposes, is carried out under substantially thesame conditions as described above.

Methods of the invention further provide a means for identifyingcellular products responsible for mediating the effect of a PTH/PTHrPreceptor expressing cell on a stem or progenitor cell. Such cellularproducts can include, for example, secreted proteins, cell-surfaceproteins, glycoproteins, lipids or steroids.

Cellular products responsible for mediating the effect of a PTH/PTHrPreceptor expressing cell on a stem or progenitor cell may regulategrowth, for example, by increasing cell division or “replication.”Accordingly, a method of identifying a cellular product that increases apopulation of stem or progenitor cells is provided, the methodcomprising the steps of:

-   -   a) contacting a cell expressing a PTH/PTHrP receptor with an        agent that activates the PTH/PTHrP receptor;    -   b) collecting proteins or mRNA encoding proteins produced by the        cell expressing a PTH/PTHrP receptor in response to the agent of        step a);    -   c) contacting a stem or progenitor cell with one or more        proteins of step b);    -   d) measuring a physiologic effect exhibited by the stem or        progenitor cell; and    -   e) isolating one or more proteins associated with the        physiologic effect,        wherein the physiologic effect comprises increased replication        of the stem or progenitor cells.

Methods of isolating individual proteins from samples containingmultiple proteins are well known in the art. For example, fractions thattest positive for a given physiologic effect (e.g., cell replication)can be further subdivided and re-tested until a sufficiently smallpopulation exists. This can be accomplished by obtaining cDNA librariesfrom activated PTH/PTHrP receptor expressing cells, dividing thelibraries into fractions and transfecting pools of cells that areultimately each co-cultured with the stem or progenitor cells andassayed for a positive response. Positive responses can be matched withone or more cell pools. cDNAs associated with a positive pool can becollected, further subdivided and re-tested until a sufficiently smallnumber of candidate cDNAs are obtained and sequenced.

Other methods of characterizing protein profiles of cells of interestare known in the art, such as Matrix-AssistedLaser-Desorption/Ionization Time-of-Flight Mass Spectrometry(“MALDI-TOF”). The presence and molecular mass of proteins in samplescan be determined using MALDI-TOF. Essentially, samples are mixed with aUV-absorbing chemical, crystallized and placed on a steel surface. Lasertreatment is used to vaporize and ionize the samples. Peptide ions arethen accelerated in an electric filed, and flight times are converted tomass values. A specialized form of MALDI-TOF, known as Surface EnhancedLaser-Desorption/Ionization Time-of-Flight Mass Spectrometry(“SELDI-TOF”) expands the approach to include the use of surface isderivatized with polypeptide binding ligands.

Characterization of gene expression profiles in response to PTH/PTHrPreceptor activation can also be used to select candidate cDNAs forfurther testing. For example, increases in gene expression followingPTH/PTHrP receptor activation can be detected by methods of subtractionhybridization. cDNAs of interest can then be cloned and expressed incells that are ultimately co-cultured with the stem or progenitor cellsand assayed for a positive response. A sufficiently small number ofcandidate cDNAs can then be obtained and sequenced.

The following description of experiments performed is exemplary andnon-limiting to the scope of the claimed invention.

EXAMPLES

Hematopoietic stem cell frequency is affected by cell autonomous,intrinsic and cell non-autonomous, extrinsic factors. The intrinsicfactors have been mapped to specific regions of the mouse genome (deHaan & van Zant, 1997, J. Exp. Med., 186:529-536) that modulate thefrequency of hematopoietic stem or restricted progenitor cells, but notboth (Morrison et al, 2002, J. Immunol., 168:635-642). Cell cycledependent kinase inhibitors (CDKIs) that are differentiation stagespecific molecular mediators of the hematopoietic stem cell (p21) orprogenitor cell (p27) pool size have been identified (Cheng et al.,2000, Nature Med., 6:1235-1240; Cheng et al., 2000, Science,287:1804-1808). However, the extent to which CDKI expression is affectedby cell extrinsic cues provided by microenvironmental stimuli remainsill defined. Overcoming CDKI imposed blockade on cell cycle entry inprogenitor cells is readily accomplished ex vivo by a number a cytokinesproduced by multiple cell types in the bone marrow and with measurablelevels in serum. In contrast, adult bone marrow derived hematopoieticstem cells are generally difficult to expand ex vivo and fewmanipulations have resulted in defined stem cell expansion in vivo.Among these are activation of the cell surface Notch1 and Wnt (Reya etal., 2003, Nature, 423:409-414; Murdoch et al., 2003, Proc. Natl. Acad.Sci. USA, 100:3422-3427) receptors (Stier et al., 2002, Blood,99:2369-2378) and overexpression of the anti-apoptotic protein, bcl-2(Weissman I et al., 2000, J. Exp. Med., 191:253-264) or the homeoboxprotein, HoxB4 (Humphries et al., 1999, Blood, 94:2605-2612). Whetherthese molecules are altered in physiologic contexts however, has notbeen defined and what cell types within the hematopoieticmicroenvironment participate in altering stem cell numbers in vivo havenot been previously characterized. The data presented here demonstratethat osteoblast specific expression of an activated receptor canmeaningfully affect both the bone and the bone marrow microenvironmentschanging bone mass and hematopoietic stem cell pool size. The datapresented here indicate that osteoblastic cells are regulatorycomponents of the hematopoietic stem cell niche in the mouse.Perturbation in the number and possibly function of these cells by PPRactivation can lead to increased stem cell numbers, apparently byincreased self-renewal. Physical interaction of the primitivehematopoietic cells with the niche is required and the Notch signalingpathway is involved. These results define the osteoblast as a regulatorof hematopoiesis and support an important in vivo interplay between boneand bone marrow.

There are several mechanisms by which PPR activation could influencehematopoiesis. Given the association of hematopoietic stem cells withendosteal surfaces in the paratrabecular space, activated PPR inducedchanges in the architecture of the bone marrow due to increased boneformation, could affect the surface area for support of stem cells.Through the expansion in physical niches capable of maintaining stemcells, a proportionate increase in the number of stem cells couldresult. Disaggregated col1-caPPR marrow stroma would not be expected toprovide a similar increase in such physical niches, however, and yet isable to increase stem cell support ex vivo. The ability of PTH toincrease LTC-IC ex vivo further argues against this explanation as it issimilarly unlikely that three dimensional niche construction is inducedin the two dimensional monolayer culture of stroma used for that assay.An alternative and more likely explanation is the ability of PPRstimulation to induce osteoblast activation thereby indirectlystimulating hematopoiesis.

To what advantage are bone forming elements coupled to hematopoiesis? Ina developmental context, mineralization of the bone and increase in bonemass occur during the second trimester when arguably the developmentalimperative is to prepare the host for post-uterine life. Withinhematopoietic tissue, this involves a shift from predominantlyerythrocyte and platelet production to generation of cellular elementsof the innate and adaptive immune system. Hematopoietic cell productiontransitions from the fetal liver as that organ acquires hepatocytepopulations and function. Movement of hematopoiesis to the bone marrowand thymus occur in relative tandem, marking changes in the lineagedifferentiation profiles of blood elements. With the shifting emphasison mature cell populations, primitive cell lineage outcomes aremodulated and stem cell cycling transitions from robust proliferation inthe fetal liver to a less vigorous cycling status in the bone marrow.Stem cells eventually acquire the relative quiescence necessary forlong-term maintenance of the mature animal (Cheng et al., 2000, Science,287:1804-1808). The translocation to bone marrow is accompanied by atransition in stem cell cycling and differentiation. The concurrence ofthese events with building the skeleton may crudely be viewed aspre-natal necessities for encountering the outside world and as roughlyneeding proportionate scaling with body mass. Failure to achieve bonemarrow hematopoiesis due to either aberrant translocalization orosteopetrosis is accompanied by severe hematopoietic defects includingin lineages not thought to be directly affected by the inducingmolecular defect (Ma Q et al., 1998, Proc. Natl. Acad. Sci. USA,95:9448-9453; Dai X M et al., 2002, Blood, 99:111-120). The link ofhematopoiesis to bone marrow appears to be important for normal bloodhomeostasis.

As hematopoiesis expands its cell repertoire, it produces populations ofcells capable of feeding back on stem cell function, modifying it inresponse to stress. To the extent that the osteoblast represents amature descendent of bone marrow tissue, it also falls into a paradigmset by other mature bone marrow derived cellular elements.Monocyte/macrophages and T cells are well known to have among theirproducts of activation, cytokines that positively and negatively affecthematopoiesis. PTHrP is increased in response to endotoxic stress inanimal models (Funk J L et al., 1997, Endocrinology, 138:2665) andactivation of osteoblasts can be increased by PTHr stimulation underconditions of stress (Ryder K D et al., 2000, Calcif. Tissu. Int.,67:241-246). Therefore, osteoblasts may be among bone marrow derivedcells capable of providing a regulating effect on hematopoiesis in thevarying post-natal environment and of providing that modulation in theimmediate microenvironment of the stem cell. The osteoblast may be amesenchymal stem cell product that can be considered a cell withpleiotropic action including feedback regulation of the cells from whichit emerges.

As well as acting as a chemotactic stimulus, SDF-1α has been shown toincrease hematopoietic stem/progenitor cell number and function throughinhibition of apoptotic pathways and promoting the cells to cycle(Lataillade et al., 2000, Blood, 95:756-768; Lataillade et al., 2002,Blood, 99:1117-1129). Hematopoietic cells which have been engineered tooverexpress the SDF-1α protein demonstrate increases in number in theadult mouse (Onai et al., 2000, Blood, 96:2074-2080).

Hematopoietic stem cells undergo a development stage-specifictranslocation during ontogeny and ultimately reside in the adult bonemarrow. Maintenance of this highly regenerative cell pool through adultlife is dependent upon the relative quiescence of stem cells. Thefollowing examples demonstrate new methods for manipulatinghematopoietic stem cells for improved therapeutic purposes. The studiesfocused on whether PTH actions on osteoblastic cells could alter theirability to support hematopoiesis. Hematopoiesis was characterized in apreviously described transgenic mouse model in which a constitutivelyactive PPR is expressed in cells of the osteoblastic lineage (Calvi etal., 2001, J. Clin. Invest., 107:277-286).

Example 1 Materials and Methods

Identification of Transgenic Mice. Mice expressing a constitutivelyactive PPR under the control of the 2.3 kb fragment of the mouse α1(I)collagen promoter (Rossert et al., 1995, J. Cell. Biol., 129:1421-1432)were previously generated (Calvi et al, 2001, J. Clin. Invest.,107:277-286). The transgene construct (FIG. 1 a) contained the 2.3 kbfragment of the mouse α1(I) collagen promoter, 1,880 bp encoding thehuman mutant PPR HKrk-H223R (Calvi et al., 2001, J. Clin. Invest.,107:277-286), and 750 bp from the pcDNAI vector (which provides a splicesequence and the consensus polyadenylation signal absent in the cDNAencoding HKrk-H223R). Genotypying and determination of number ofinsertion sites of the transgene were performed as described (Calvi etal., 2001, J. Clin. Invest., 107:277-286). All studies performed wereapproved by the institutional animal care committee.

Transgene expression. To confirm transgene expression by in situhybridization, a 596 bp probe (DT7) was generated by PCR amplificationof pcDNAI vector sequence in the transgene construct using the reverseprimer A1 (5′-TAATACGACTCACTATAGGGCGATAAACAAGTTAACAACAACAAT-3′ SEQ IDNO:2) and the forward primer S2 (5′-CTTTGTGAAGGAACCTTACT-3′ SEQ ID NO:3)(Calvi et al., 2001, J. Clin. Invest., 107:277-286). The A1 reverseprimer sequence includes also the T7 RNA polymerase binding site. ThePCR conditions were as follows: 94° C. for 1 min, 58° C. for 45 sec, 72°C. for 1 min, and an additional 10 min at 72° C. at the end of the 45cycles. In situ hybridizations were performed as described (Calvi etal., 2001, J. Clin. Invest., 107:277-286) using a complementary³⁵S-labeled riboprobe transcribed from the DT7 PCR product in order todetect expression of the transgene mRNA in stromal cells.

Sample preparation and histologic analysis. For histologic analysis,transgenic mice and sex-matched wild type littermates were sacrificed bycervical dislocation at 12 weeks of age. Tissues from transgenic andwild type littermates were fixed and stored as described (Calvi et al.,2001, J. Clin. Invest., 107:277-286). Hind limbs were decalcified (Calviet al., 2001, J. Clin. Invest., 107:277-286), and paraffin blocks wereprepared by standard histological procedures.

For immunohistochemistry, decalcified sections from wild-type andtransgenic mice were stained with the anti-IL-6 gAb M-19 (1:100dilution), the anti-SCF gAb G-19 (1:100 dilution), the anti-SDF-1 gAbC-19 (1:50 dilution), the anti-Osteopontin gAb P-18 (1:200 dilution),and the anti-Jagged1 rAb H-114 (1:100 dilution) (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The immunohistochemicalstaining was performed using a biotinylated rabbit anti goat or goatanti rabbit secondary Ab (Vector Labs, Burlingame, Calif.), HorseradishPeroxidase-Conjugated Streptavidin (Jackson Immuno Research, West Grove,Pa.), and AEC Chromogen (Biocare Medical, Walnut Creek, Calif.), or theVector ABC Alkaline Phosphatase Kit (Vector Labs, Burlingame, Calif.).Slides were counterstained with Mayer's hematoxylin.

Cytologic analysis. For cytologic analysis, hind limbs were dissectedfrom euthanized wild type and transgenic littermates and cellspreparations were obtained by flushing the long bones with α-MEM with10% fetal calf serum (Gibco) and 1% penicillin/streptomycin. Cells werethen cultured in tissue culture flasks at an initial concentration of5×10⁶ cells/ml. Medium was changed every three days for two weeks oruntil stromal layers became confluent. Adherent cells were thentrypsinized and plated at a concentration of 10⁵ cells/ml in multiwellchambers for 7, 14, 28 days and medium was changed every 3 days. For insitu hybridization, cells were rinsed with PBS three times and thenfixed for 1 hr at room temperature with 3.7% PBS buffered formaldehyde.For immunohistochemistry, cells were rinsed with TBS.Ca (1 mM CaCl₂, 50mM Tris/HCl pH 7.4, 150 mM NaCl) four times and fixed for 1 minute witha 1:1 solution of acetone and methanol at room temperature.

Immunocytochemistry. Immunocytochemical staining was performed onAcetone:methanol fixed stromal cells. Cells grown in multiwell plateswere incubated with anti-SDF-1 goat polyclonal Ab (Santa CruzBiotechnology, Santa Cruz, Calif.), 1:50 dilution, for 60 minutes atroom temperature, and for 45 minutes with a fluorescein-conjugatedsecondary antibody. Cells were counterstained with Evan's blue.Coverslips were mounted with Vectashield containing DAPI (VectorLaboratories, Burlingame, Calif.), and slides were examined using afluorescent microscope with the appropriate filter.

Preparation of bone marrow stromal layers. Mice were euthanized by CO₂asphyxiation, following which the femurs and tibias were removed andflushed with long-term culture medium (α-MEM with 12.5% horse serum,12.5% fetal bovine serum, 0.2 mM i-Inositol, 20 μM folic acid, 10⁻⁴ M2-mercaptoethanol, 2 mM L-glutamine and 10⁻⁶ M hydrocortisone; M5300Stem Cell Technologies). Mononuclear cells were then cultured in tissueculture flasks at an initial concentration of 5×10⁶ cells/ml. Medium waschanged every three days for two weeks or until stromal layers becameconfluent.

Flow cytometric analysis. Bone marrow mononuclear cells were isolated asdescribed above. Single cell suspensions were then stained withbiotinylated lineage antibodies (CD3, CD4, CD8, Ter119, Gr-1, Mac-1 andB220) and phycoerythrin conjugated anti-Sca-1 and anti-c-Kit(Pharmingen, San Diego, Calif.). Cells were then labeled with asecondary fluorescein isothiocyanante conjugated streptavidin andanalyzed on a FACScalibur cytometer (Becton Dickinson and Co., FranklinLakes, N.J.) using Cell Quest software. To assess cell cycle in theprimitive population, bone-marrow mononuclear cells (BM MNCs) werestained with lineage antibodies, anti-Sca-1, PyroninY (RNA dye) andHoechst 33342 (DNA dye) as described (Cheng et al., 2000, Nature Med.,6:1235-1240). For intracellular NICD staining, lin⁻Sca-1⁺c-Kit⁺ cellswere permeabilized using the Fix and Perm Cell Permeabilization Kit(Caltag) according to manufacturer's instructions and incubated with 1μg of anti-NICD antibody. A secondary goat-anti mouse antibody was thenused to detect the anti-NICD.

Colony forming unit assay. Mononuclear cells were isolated from the bonemarrow and cultured at 10⁴ cells/ml in the following medium: 0.9%methylcellulose, 15% FBS, 1% BSA, 10 μg/ml rh insulin, 200 μg/ml humantransferrin, 10⁻⁴ M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml rmSCF,10 ng/ml rmIL-3, 10 ng/ml rhIL-6 and 3 units/ml rhEpo (M3434; Stem CellTechnologies, Vancouver, Canada). At day 10, the total number ofcolonies were counted and reported as total CFU-Cs.

Long-term culture initiating cell assay. Murine bone marrow stromalcells from confluent cultures were irradiated (15Gy) and plated at aconcentration of 20,000 cells/well in 96-well plates in long-termculture medium. Cells were then seeded into the plates in two-foldserial limiting dilutions and cultured at 33° C./5% CO₂ in a humidifiedatmosphere. Cultures were maintained for 5 weeks, changing half of themedium in the wells weekly. Following this, the medium was replaced withmethylcellulose containing medium supplemented with recombinantcytokines as described above, then scored for colony growth ten daysfollowing the addition of the medium.

In vitro treatment with PTH. LTC-IC assays were performed usingwild-type stroma and hematopoietic cells. Rat PTH (1-34) (Bachem,Torrance, Calif.) or vehicle was added to each media change eitherduring stroma establishment and/or during culture maintenance to a finalconcentration of 10⁻⁷ M. Medium was changed every three days for twoweeks or until stromal layers became confluent. For alkaline phosphatasestaining, primary mononuclear cells obtained as described above fromwild-type and or transgenic littermates were then cultured in 24-wellplates at an initial concentration of 5×10⁶ cells/ml. At 10 or 14 daysafter seeding, medium was suctioned off, and the adherent cells weregently rinsed twice in PBS. After fixation in 10% Neutral FormalinBuffer for 30 minutes at room temperature, alkaline phosphatase activitywas determined histochemically by incubation for 45 minutes at RT with amixture of 0.1 mg/ml naphthol AS-MX phosphate (Sigma), 0.5%N,N-dimethylformamide, 0.6 mg/ml red violet LB salt (Sigma) in 0.1 MTris-HCl (pH 8.5). Alkaline phosphatase positive cells were counted atday 10 of culture, when the cultures were subconfluent and individualcells could be identified. For inhibition of γ-secretase activity, 30 μMof γ-secretase inhibitor II (Calbiochem) dissolved in DMSO was added tothe long-term culture medium and LTC-IC assays were performed asdescribed. For non-contact LTC-ICs, bone marrow stromal cell layers wereplated into 96-well plates as described. Tissue culture inserts with a0.2 μm pore size membrane (Nunc, Naperville, Ill.) were placed in thewells and bone marrow cells were seeded into the culture inserts.

In vivo PTH administration. For PTH administration, 6-8 week old wildtype C57/B male mice were used. Rat PTH (1-34) (80 μg/Kg of body weight)was injected intraperitoneally 5×/week for 4 weeks (n=5). Control mice(n=4) were injected with an equivalent volume of vehicle. At the end ofthe treatment period, ionized serum calcium was measured by theCiba/Corning 634 Ca⁺⁺/pH analyzer, and, after euthanasia, the hind limbsand forelimbs were dissected and utilized for cytologic and histologicanalysis.

SDF-1 ELISA. The amount of SDF-1 released in the cell culturesupernatant was estimated by ELISA. SDF-1 concentration was measured inconditioned media from subconfluent primary stromal cells cultures fromtransgenic and wild type littermates using the Quantikine SDF-1Immunoassay (R&D Systems, Inc, Minneapolis, Minn.).

Bone Marrow Transplantation. For the competitive transplant studies,4×10⁵ BM MNCs, obtained from CD45.1⁺ B6.SJL (Jackson Laboratories, BarHarbor, Me.) mice were mixed with 2×10⁵ cells from mock injected or PTHinjected CD45.2⁺ C57B1/6 mice. Recipient B6.SJL mice that had beenlethally irradiated 24 hours previously with 10Gy of radiation (¹³⁷Cssource) were injected with cells. After 6 weeks, the mice wereeuthanized with CO₂, the BM was removed and flushed with fullysupplemented Iscove's Medium. The relative contribution of engraftmentfrom the different cell sources was assessed by flow cytometry utilizinganti-CD45.1 and anti-CD45.2 antibodies (Pharmingen, San Diego, Calif.).To assess the effects of PTH administration post transplantation,recipient C57B1/6 mice were lethally irradiated and then injected with2×10⁵ BM MNCs from a donor B6.SJL mouse. Twenty-four hours following theinjection of the cells, mice were injected with PTH or mock injected asdescribed above for four weeks.

Statistical analysis. Results are expressed as mean+/−s.e.m. Data wereanalyzed using the unpaired two-tailed Student's t test as appropriatefor the data set. P<0.05 was considered significant.

Example 2 Transgenic Mouse Experiments

Transgenic mice expressing a constitutively active PPR in cells of theosteoblastic lineage have bone marrow fibrosis and anemia. At 2 and 12weeks of age, the long bones of the col1-mutPPR mice were characterizedby abundant trabeculae and marrow fibrosis. At 12 weeks of age, the longbones of the col1-caPPR mice were histologically examined anddemonstrated abundant trabeculae with reduced volume of the marrow spacein the metaphyseal area. Given the modest contribution of themetaphyseal area to the total marrow space of the long bones, themagnitude of the reduction in the total bone marrow space in the longbones of the adult transgenic mice was minimal. There was an expansionof the trabecular osteoblastic population as defined by staining withosteocalcin, alkaline phosphatase, collagen type I, osteopontin andMMP-13 (Calvi et al., 2001, J. Clin. Invest., 107:277-286).Hematopoietic cells were found in small regions between trabeculae andfew adipocytes were seen. Transgenic mice had mild anemia (hematocrit,wild-type, n=5: 41+/−0.2%; transgenic, n=4: 35.9+/−0.6%, P<0.005), afinding also noted in humans with severe primary hyperparathyroidism(Kotzmann et al., 1997, Horm. Metab. Res., 29:387-392; Sikole, 2000,Med. Hypoteses, 54:236-238). This particular phenotype suggests thatconstitutive activation of the PPR in cells of the osteoblast lineagemay alter normal hematopoiesis by affecting the stromal cell population.Bone marrow stromal cells from transgenic mice express the mRNA of thehuman mutant PPR in culture.

Transgenic mice have an increased number of hematopoietic stem cells inthe bone marrow. To elucidate the impact of enhanced activity and numberof osteoblasts on hematopoietic stem cells in the transgenic mice, thefrequency of hematopoietic stem cells in the bone marrow was firstexamined by flow cytometry. Analysis of the frequency of the Sca-1⁺lin⁻subpopulation of cells from the total bone marrow mononuclear cellsdemonstrated that the transgenic mice had a significant increase in thenumber of candidate stem cells (P=<0.01, FIG. 2 a). This proportionateincrease had a correspondent increase in the absolute number (meanabsolute number per hind limb, wild-type: 32,500+/−8,000 versustransgenic: 65,700+/−7,500). To determine if this corresponded to afunctional phenotype, a quantitative, limiting dilution long-termculture initiating-cell (LTC-IC) assay was used that linearly correlateswith in vivo hematopoietic stem cell (HSC) function (Ploemacher et al.,1991, Blood, 78:2527-2533). The hematopoietic stem cell frequency wasexamined using the functional measurement of LTC-IC frequency in thelin⁻ fraction of bone marrow mononuclear cells. This confirmed anincrease of approximately equivalent magnitude in the frequency ofLTC-ICs in the transgenic animals (P=<0.0001, FIG. 2 b). The magnitudeof increase was comparable to the increase seen in immunophenotypicallydefined primitive cells. As this increase in stem cell frequency couldhave arisen from an alteration in the cell cycle profile in thetransgenic animals, the proportions of Sca-1⁺lin⁻ cells which were inthe G₀ versus G₁ phase were analyzed next. No differences were observedbetween the transgenic and wild-type mice (P=0.768, FIG. 2 c).Similarly, measurement of the frequency of hematopoietic progenitorcells using the CFU-C assay demonstrated no difference between thetransgenic and wild-type animals (P=0.573; FIG. 2 d). These datademonstrate the specificity of the expansion to be at the hematopoieticstem cell level. In particular these data demonstrate that cellexpansion was not global across differentiated subsets, but was notablyrestricted to primitive cells.

PTH action on stromal cells through the PPR is sufficient to increasenumbers of hematopoietic stem cells in vitro. As the transgenic mice hadan increased frequency of hematopoietic stem cells, the mechanism forthis enhancement was investigated. Evaluating the ability of bone marrowstromal cells to support LTC-ICs, it was found that stromal cellsderived from the transgenic mice demonstrated enhanced LTC-IC supportcompared with stromal cells from wild-type animals (P=<0.005, FIG. 3 a).Therefore the increased number of primitive cells in the col1-caPPR micewas stroma-determined and was independent of the hematopoietic cellgenotype. Due to the transgenic mice having a constitutively activePTH/PTHrP receptor, it was then determined whether or not the additionof exogenous PTH could mimic the previous observations. In theseexperiments, the stromal cell population was expanded in the presence ofPTH, or the LTC-IC assay was performed with PTH in the long-term medium.It was found that the presence of PTH during the expansion of thestromal population from the bone marrow enhanced the ability of thestroma to support LTC-ICs.

Example 3 Transgenic Cell Experiments

PPR transgenic osteoblastic cells highly express IL-6, SCF, and SDF-1.

Immunohistochemistry was used to assess levels of Interleukin 6 (IL-6),kit ligand or Stem Cell Factor (SCF) and Stroma-derived Factor 1 (SDF-1)in the transgenic cells among the metaphyseal trabeculae. These cellshave been previously shown by in situ hybridization to be aheterogeneous population of osteoblastic cells (Calvi et al., 2001), andimmunohistochemistry for Osteopontin, a marker of osteoblastic cells,confirmed these data. In wild-type animals, only a few osteoblasticcells express these factors. In contrast, high levels of IL-6 weredetected heterogeneously in the osteoblastic cells of transgenicanimals. Expression of SDF-1 was diffuse, while SCF was present at highlevels mainly in the more mature cells lining the trabeculae. To addresswhether diffusible cytokines could account for the effect on primitivecells, LTC-ICs were performed with a semi-permeable membrane separatingfeeder cells from BM MNCs (non-contact cultures) and noted abolition ofbenefit from the activated PPR(P=0.982, FIG. 4). These data indicate therequirement for cell-cell contact or direct primitive hematopoietic cellinteraction with a niche cell or matrix component. SCF may be membranebound as well as freely secreted. However, other candidate membranerestricted mediators of stem cell expansion were investigated.

Transgenic osteoblastic cells produce high levels of the Notch ligand,Jagged1. The Notch signalling pathway, which regulates cell-fatespecification in a wide variety of systems (Artavanis-Tsakonas et al,1999, Science, 284:770-776), is thought to affect HSC self-renewal(Stier et al., 2002, Blood, 99:2369-2378; Varnum-Finney et al., 2003,Blood, 101:1784-1789; Varnum-Finney et al., 2000, Nat. Med.,6:1278-1281; Karanu et al., 2000, J. Exp. Med., 192:1365-1372; Karanu etal., 2001, Blood, 97:1960-1967). Manipulation of Notch signalling hasbeen shown to increase stem cell numbers without expanding mature cells(Stier et al., 2002, Blood, 99:2369-2378; Karanu et al., 2000, J. Exp.Med., 192:1365-1372). Further, the Notch ligand Jagged1 has been shownto be expressed by marrow stromal cells (Karanu et al., 2000, J. Exp.Med., 192:1365-1372; Li et al., 1998, Immunity, 8:43-55) as well asmurine osteoblasts (Pereira et al., 2002, J. Cell Biochem., 85:252-258).Notch and cytokine induced signalling pathways have been shown to have acombinatorial effect in regulating hematopoietic cell fate(Varnum-Finney et al., 2003, Blood, 101:1784-1789). It was thereforeinvestigated whether Jagged1 protein levels were altered in the marrowof transgenic mice and observed by immunohistochemistry that overalllevels of Jagged1 were dramatically increased. The cells expressing theJagged1 were osteoblastic, as shown by their morphologic characteristicsand staining with anti-Osteopontin antibody. To examine whether thehematopoietic stem cells responded to the increased expression ofJagged1 in the transgenic animals, the level of the Notch IntracellularDomain (NICD) was assessed in the lin⁻Sca-1⁺c-Kit⁺ HSCs from wild-typeand transgenic mice. The anti-NICD antibody has previously been shown topreferentially detect the activated intracellular form of Notch1(Huppert et al., 2000, Nature, 405:966-970). Whereas wild-type mice hadminimal staining for the NICD compared with isotype controls,lin⁻Sca-1⁺c-Kit⁺ cells from transgenic mice had a notable increase inthe level of NICD (FIG. 3 b). These data suggest a model in whichactivation of the PPR in the osteoblastic population increases theirnumber and their overall production of Jagged1. This in turn mayactivate Notch1 on primitive hematopoietic cells resulting in expansionof the primitive cell compartment.

Example 4 In vitro PTH Administration

PTH treatment in vitro reproduces the coil-caPPR effect. Since thecol1-caPPR mice represented a genetic means of activating a receptorthat could also be activated by endogenous ligand, it was next testedwhether the effects of col1-caPPR stroma could be recapitulated throughexposure of wild type stroma to PTH. LTC-IC assays were performed usingC57B1/6 stroma expanded in vitro in the presence or absence of PTH,after which hematopoietic cells were introduced to the stroma in thepresence or absence of PTH. When stroma was grown in medium containingPTH, it closely resembled the LTC-IC results using the col1-caPPRstroma, increasing LTC-IC (P=0.004, FIG. 5 a). Of note, the effect wasnot seen using stromal cells that were expanded in the absence of PTH,or when PTH was added at the same time as the hematopoietic cells,suggesting an effect on the composition or activity of the stroma as itmatures in vitro. To assess whether there was an increase in theosteoblastic cell number in the stromal cell cultures treated with PTH,alkaline phosphatase staining of primary murine stromal cell culturestreated with vehicle or PTH were performed. After 14 days, the cultureswere confluent and heterogeneous, and there was an increase in alkalinepositive cells in the PTH-treated cultures (FIG. 5 b), verifying thatactivation of PPR induces an increase in the number of osteoblasticcells. To further assess whether the effects of PPR activation onprimitive hematopoietic cells were due to Notch pathway activation, longterm co-cultures in the presence or absence of a γ-secretase inhibitorcapable of blocking Notch1 cleavage (Wolfe et al., 1999, Biochemistry,38:4720-4727) were performed. Addition of the inhibitor reduced thesupportive capacity of PTH treated stroma to baseline levels (FIG. 5 c).Therefore, Notch1 activation is necessary for osteoblastic cell inducedincreases in primitive hematopoietic cells. Taken together, theseresults further support the model that PPR activation can increaseosteoblastic cells resulting in Notch1 mediated expansion of primitivehematopoietic cells.

Example 5 In Vivo PTH Administration

Intermittent PTH treatment of normal mice. As treatment of stromal cellswith PTH led to an increase in the ability of the stromal cellpopulation to support hematopoietic stem cells, it was investigatedwhether these effects could be recapitulated in vivo. Wild-type C57B1/6mice were injected daily with PTH using an intermittent dosing scheduleknown to increase osteoblasts, and the frequencies of LTC-ICs in thebone marrow were measured. Whereas a two week PTH treatment period didnot result in any significant increase in the hematopoietic stem cellpopulation, treatment of mice for four weeks with PTH resulted in asignificant increase in the hematopoietic stem cells over mock injectedmice. By four weeks, a significant difference was noted with PTH treatedmice having a higher frequency and absolute number of lin⁻Sca-1⁺c-Kit⁺compared with mock treated controls (P=<0.01, FIG. 5 d). Further, thelimit dilution LTC-IC assay demonstrated an increase in stem-like cells(P=<0.005, FIG. 5 e). To further define that functional stem cells wereincreased after PTH treatment, an in vivo assay of competitivetransplantation into secondary recipients was used and a >2-foldincrease in HSCs was documented (P=<0.05, FIG. 5 f). These data provideevidence for an increase in HSCs induced by PTH and also serve tovalidate the reasonable comparability of the in vitro and in vivo assaysused in these studies. Consistent with observations in the transgenicanimals, PTH treatment did not affect the level of hematopoieticprogenitors as assessed by CFU-C assay (P=0.780, FIG. 5 g). Therefore,pharmacological activation of PPR increased stem cell number, butappeared to do so without a broad hematopoietic cell expansion. Thesedata are most consistent with HSC expansion by enhanced self-renewal, aphenomenon known to result from Notch activation. Of note, there was noevidence of hypercalcemia by serum calcium measurements of the PTHtreated animals.

PTH administration in vivo following bone marrow transplantation.Assessing whether PPR stimulation could affect models relevant to theclinical use of stem cells in humans, the impact of PTH administrationon animals undergoing myeloablation and bone marrow transplantation wasassessed. Limiting numbers of bone marrow derived donor cells were usedto mimic a setting of therapeutic need. Survival rate at 30 days incontrol mice receiving mock injections after bone marrow transplantationwas 40%. In sharp contrast, animals receiving pulse dosing of PTH hadmarkedly improved outcomes with 100% survival (FIG. 6).

Example 6 Preventing Chemotherapic Damage with PTH

PTH provides a protective effect during a course of G-CSF andcyclophosphamide therapy. Cyclophosphamide is used as a chemotherapeuticagent in the treatment of hematological malignancies. Due to themyelotoxic effects of cyclophosphamide on the bone marrow, granulocytecolony-stimulating factor (“G-CSF”) is typically administered to augmentthe recovery from the chemotherapy-induced neutropenia. However,treatment with G-CSF following administration of cyclophosphamide isknown to reduce the hematopoietic stem cell (HSC) sub-population ofcells in the bone marrow. The protective effect of PTH administrationduring a course of G-CSF and cyclophosphamide therapy was observed asdescribed herein.

PTH administration in vivo. Wild-type C57B1/6 mice were treated with 5mg of cyclophosphamide (Day 1). The day following injection ofcyclophosphamide, mice received no treatment or were treated for 8 dayswith G-CSF (5 μg/day), 11 days with PTH (80 μg/kg/day) or a combinationof G-CSF plus PTH. Mice that received a combination of both G-CSF andPTH received G-CSF treatment for 8 days and PTH treatment for 11 days.Following this treatment protocol, mice were injected withcyclophosphamide on Day 15 and treated with G-CSF, PTH or G-CSF and PTHas described above. The treatment protocol was then repeated for twosubsequent cycles, as outlined in FIG. 7.

Complete blood count analysis. During the course of the four treatmentcycles, peripheral blood complete blood count (CBC) analysis wasperformed every 2 to 3 days. This analysis involved the collection of100 μl of peripheral blood from the tail vein of the mice, which wassubsequently analyzed on a HEMAVET 850FS (Drew Scientific). To avoid anydeleterious effects from repeated tail bleeds, each individual mouse wasonly bled once per week.

Assays for hematopoietic stem cells. At the end of the four-cycletreatment with cyclophosphamide and G-CSF/PTH, the bone marrow wasremoved from the treated mice and competitive transplants into B6.SJLmice were performed to measure the maintenance of the HSC pool in thebone marrow. This involved mixing 5×10⁵ BM cells from the treatedC57B1/6 mice (CD45.2) with 2.5×10⁵ BM cells from the B6.SJL mice(CD45.1). These cells were then injected into lethally irradiated B6.SJLmice. Eighteen weeks following injection of the cells, peripheral bloodwas collected from the mice and the relative contribution of the HSCsfrom the treated animals was measured by flow cytometry for the CD45.2⁺cells. To assess the ability of the HSCs to be mobilized into theperipheral circulation, one week following treatment mice were mobilizedwith G-CSF (5 μg/day) for 5 days. Peripheral blood (300 μl) wascollected from these mice and mixed with 2.5×10⁵ BM cells from a B6.SJLmouse. These cells were injected into a lethally irradiated B6.SJL host.Eighteen weeks following injection of the cells, peripheral blood wascollected from the mice and the relative contribution of the HSCs fromthe treated animals was measured by flow cytometry for the CD45.2⁺cells.

Administration of PTH does not alter the hematopoietic responsefollowing chemotherapy, with or without G-CSF support. Analysis of theperipheral blood in terms of white blood count (WBC), neutrophils count(NE), hemoglobin concentration (Hb) and platelet count (Plt) during the8-week chemotherapy treatment period is shown in FIG. 7, A-D. Mice thatreceived G-CSF demonstrated significant increases in their WBC and NEcounts following chemotherapy that was not altered by the addition ofPTH treatment. Similarly, mice that did not receive G-CSF demonstratedno differences in their hematological response to chemotherapy whetherthey received PTH or not. The Hb and Plt responses were notsignificantly different between any of the treatment groups.

Administration of parathyroid hormone (PTH) results in a preservation ofthe HSC pool following chemotherapy. Analysis of the HSC pool in thebone marrow following chemotherapy demonstrated that PTH treatmentincreased the HSC pool in the non-G-CSF treated animals followingmyelotoxic chemotherapy (FIG. 7E). In the animals treated with G-CSFalone, there was a significant depletion of the HSC pool, as has beendescribed by others. However, the concurrent treatment with PTH led toretention of the HSC pool (FIG. 7E). Analysis of the mobilization of theHSCs into the peripheral circulation with G-CSF demonstrated that inmice that did not receive supportive G-CSF therapy during themyeloablative chemotherapy there was mobilization of HSCs into thecirculation, which was increased with prior PTH treatment (FIG. 7F).However, mice that received supportive growth factor therapy aloneshowed little to no mobilization of the HSCs into the peripheralcirculation, which was partially reversed by the concurrent treatmentwith PTH (FIG. 7F).

Taken together, these studies demonstrate that targeting the niche canprotect, or even expand the HSC pool in the bone marrow duringmyelotoxic chemotherapy. This is especially evident when growth factorsupportive therapy is used in conjunction with chemotherapy. Theseresults illustrate the usefulness of PTH therapy in protection ofhematopoietic stem cells during myelotoxic chemotherapy.

Example 7 Kinetics of Leukemia Cell Engraftment, Growth and Maturation

The kinetics of leukemia cell engraftment, growth, and maturation werestudied in normal mice. In an effort to analyze the growth and fate ofC-1498 leukemia cells transplanted into mice, the cells were transducedwith a retrovirus expressing GFP (C-1498/GFP). This allows easy trackingof these cells in vivo using GFP as a marker (FIG. 9 a). The acutemyelogenous leukemia cell line C-1498, a spontaneously-arising acutemyelomonocytic leukemia derived from C57/BL6 (H2-b) mice, is easilytransplantable, can be cultured in vitro, and infected withretroviruses.

As shown in FIG. 9 b, upon transplantation of 50,000 C-1498/GFP cellsinto mice, the number of GFP-positive cells in the marrow was 2% at 2days, 8% by 7 days, and 12% by 18 days. At 7 days, there were no blastsin the periphery. However, at 15-18 days, blasts began to reliablyappear in peripheral blood. At 22 days, most mice were moribund.

Attention was subsequently directed to determining how activation of thestem cell niche with PTH might affect leukemia growth and maturation. Toinvestigate the kinetics of leukemia outgrowth in “niche-stimulated”animals, animals were pre-stimulated with PTH at 80 μg/kg/day (versussaline alone) for one month. Mice were then lethally irradiated at 10Gray, and then transplanted with 50,000 leukemia cells (GFP+) and 1×10⁶normal bone marrow cells (GFP−) (FIG. 10 a). At 15 dayspost-transplantation, bone marrow was harvested, and, then, GFP-positivecells were scored as percentage and in absolute number. Since theabsolute number of total cells was not statistically different betweenboth groups, only the percentage figures are shown in FIG. 10 b, leftpanel. Of note, in 3 of 3 experiments, the number of GFP-positiveleukemia initiating cells was markedly reduced in the PTH treatedanimals.

To obtain a histological correlate of these results, 10 micron bonesections were cut from transplanted mock-treated and PTH-treatedanimals. In mock-treated animals, osteoblasts and osteoprogenitors,identified at PTH-receptor positive cells, were found lining theendostial surfaces of bone. In contrast, in PTH-treated animals, PTH-rpositive cells had migrated extensively into the marrow. Concomitantly,while GFP-positive leukemia cells were found in mock-treated animals(particularly along the bone surfaces), GFP-positive cells were markedlyreduced in PTH-treated animals (FIG. 10 b, right panel).

The ability of PTH treatment to alter the relative proportion of normalto leukemia initiating stem cells may involve several mechanisms. First,the cells may compete for niche spaces that are different and the PTH‘expanded’ niches may not have a proportionate likelihood of supportingleukemic and normal stem cells. The ability to modify the relativeabundance of normal to leukemic cells by increasing the ratio of normalto leukemic cells suggests that such a mechanism may apply. Differentialproduction of normal cells over leukemic cells in a PTH treated animaldid not seem to be the case as kinetics of normal hematopoiosis did notchange (FIG. 11). However, differential sensitivity to an inhibitingsignal may be a potential mechanism as was further tested.

Since PTH did not directly affect leukemic cells in vitro, theosteoblast-leukemic cell interaction was further characterized.Osteoblasts and C 1498/GFP leukemia cells were co-cultured. The schemefor this experiment is shown in FIG. 12 a. Bone marrow was harvestedfrom wild-type and Col A-PPR* mice, and the total cellular content wascultured for 10 days in vitro. After 10 days of culture, cells weredissociated into single cell suspension. Osteoblasts express the PTHreceptor, but not CD45, a universal marker for hematopoietic cells. Topurify osteoblasts from the in vitro cultures, CD 45−, PTH receptor+fraction was isolated by FACS (FIG. 12 b). This population wassubsequently stained by Alkaline Phosphatase and shown to be Alk+,indicating that these were purified osteoblasts (FIG. 12 c).

After the isolation of osteoblasts, osteoblasts and C1498/GFP leukemiacells were co-cultured (FIG. 12 d). 3 days after co-culture, thefraction of GFP+positive cells and total cells was counted by FACSanalysis again (FIG. 12 e). Of note, there was a significant reductionof leukemic cells in the osteoblast co-cultures compared to the controlcultures (fibroblast alone). In addition, leukemic clone sizes inosteoblast cultures were found to be smaller (1-2 cells), while clonesizes in fibroblast cultures were 5-8 cells, suggesting that leukemiccells have reduced capacity to expand in the presence of osteoblasts.

To further investigate the phenomenon of osteoblast-mediated leukemiacell inhibition (as demonstrated by the preliminary in vitro experimentsdescribed herein), a candidate molecule approach was taken. Osteopontin(OPN), a molecule secreted by osteoblasts, is a key regulator of thenormal hematopoietic stem cell niche. In particular, OPN appears tolimit the number of early primitive hematopoietic cells (Stier, S. etal. (2005) J. Exp. Med. 201(11):1781-91).

To investigate the role of OPN as a candidate molecule, it was exploredwhether recombinant OPN is sufficient to reproduce the growth inhibitionof leukemia cells in vitro. To this end, C-1498/GFP cells were culturedin wells with either (a) no OPN, (b) with 5 and 10 ug/ml of OPN. Ofnote, OPN was found sufficient to reproduce the inhibition of leukemiacell growth in this system (FIG. 13 b). Staining with Annexin furtherrevealed that the effect was due to OPN mediated growth inhibitionrather than accelerated apoptosis (FIG. 13 a). It was thereforeconcluded that recombinant osteopontin is sufficient to limit leukemiccell expansion. The relative ability of OPN to limit primitive cellexpansion was greater in leukemic compared to normal cells. Given thatPTH increases osteoblast production of OPN, it is likely that OPNparticipates in the preferential support of normal over leukemic cellsin vivo with PTH stimulation of the stem cell niche.

In view of the results presented herein, it was concluded that PTH/PTHrPreceptor stimulation with PTH and its analogues can affect the relativebalance of normal and malignant cell function, thereby changing thephenotype of the malignancy to the benefit of the patient. Accordingly,leukemic and pre-leukemic conditions (e.g., myelodysplastic syndrome)characterized by the progressive domination of abnormal cells can beameliorated or eradicated by that PTH/PTHrP receptor stimulation. Giventhat PTH receptor is on many stromal cell components of many tissues, itmay be that PTH stimulation is capable of altering normal and malignantcell ratios in many tumor types.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1-107. (canceled)
 108. A method for treating a subject having or at riskof having leukemia, the method comprising: administering to the subjecta PTH, a PTH analogue, or a PTH/PTHrP receptor agonist in an amounteffective to increase the amount of normal hematopoietic stem andprogenitor cells; and decreasing the amount of leukemic or pre-leukemiccells, thereby treating a subject having or at risk of having leukemia.109. The method of claim 108, wherein the leukemia is chronic.
 110. Themethod of claim 109, wherein the chronic leukemia is chronic myeloid,chronic myelogenous or chronic granulocytic leukemia.
 111. The method ofclaim 108, wherein the leukemia is acute.
 112. The method of claim 111,wherein the acute leukemia is acute lymphoblastic leukemia or acutenonlymphoblastic leukemia. 113-122. (canceled)